Methylation Assay Research Articles

Regulation and tumor-suppressive function of the miR-379/miR-656 (C14MC) cluster in cervical cancer.

Cervical cancer (CC) is a key contributor to cancer-related mortality in several countries. The identification of molecular markers and the underlying mechanism may help improve CC management. We studied the regulation and biological function of the chromosome 14 microRNA cluster (C14MC; miR-379/miR-656) in CC. Most C14MC members exhibited considerably lower expression in CC tissues and cell lines in The Cancer Genome Atlas (TCGA) cervical squamous cell carcinoma and endocervical adenocarcinoma patient cohorts. Bisulfite Sanger sequencing revealed hypermethylation of the C14MC promoter in CC tissues and cell lines. 5-aza-2 deoxy cytidine treatment reactivated expression of the C14MC members. We demonstrated that C14MC is a methylation-regulated miRNA cluster via artificial methylation and luciferase reporter assays. C14MC downregulation correlated with poor overall survival and may promote metastasis. C14MC activation via the lentiviral-based CRISPRa approach inhibited growth, proliferation, migration, and invasion; enhanced G2/M arrest; and induced senescence. Post-transcriptional regulatory network analysis of C14MC transcriptomic data revealed enrichment of key cancer-related pathways, such as metabolism, the cell cycle, and phosphatidylinositol 3-kinase (PI3K)-AKT signaling. Reduced cell proliferation, growth, migration, invasion, and senescence correlated with the downregulation of active AKT, MYC, and cyclin E1 (CCNE1) and the overexpression of p16, p21, and p27. We showed that C14MC miRNA activation increases reactive oxygen species (ROS) levels, intracellular Ca2+ levels, and lipid peroxidation rates, and inhibits epithelial-mesenchymal transition (EMT). C14MC targets pyruvate dehydrogenase kinase-3 (PDK3) according to the luciferase reporter assay. PDK3 is overexpressed in CC and is inversely correlated with C14MC. Both miR-494-mimic transfection and C14MC activation inhibited PDK3 expression. Reduced glucose uptake and lactate production, and upregulation of PDK3 upon C14MC activation suggest the potential role of these proteins in metabolic reprogramming. Finally, we showed that C14MC activation may inhibit EMT signaling. Thus, C14MC is a tumor-suppressive and methylation-regulated miRNA cluster in CC. Reactivation of C14MC can be useful in the management of CC.

14-Week exercise training modifies the DNA methylation levels at gene sites in non-Alzheimer's disease women aged 50 to 70 years

Exercise training emerges as a key strategy in lifestyle modification, capable of reducing the risk of developing Alzheimer's disease (AD) due to risk factors such as age, family history, genetics and low level of education associated with AD. We aim to analyze the effect of a 14-week combined exercise training (CT) on the methylation of genes associated with AD in non-alzheimer's disease women. CT sessions lasted 60 min, occurring three times a week for 14 weeks. Forty non-Alzheimer's disease women aged 50 to 70 years (60.7 ± 4.1 years) with a mean height of 1.6 ± 0.1 m, mean weight of 73.12 ± 9.0 kg and a mean body mass index of 29.69 ± 3.5 kg/m2, underwent two physical assessments: pre and post the 14 weeks. DNA methylation assays utilized the EPIC Infinium Methylation BeadChip from Illumina. We observed that 14 weeks of CT led to reductions in systolic (p = 0.001) and diastolic (p = 0.017) blood pressure and improved motor skills post-intervention. Among 25 genes linked to AD, CT induced differentially methylated sites in 12 genes, predominantly showing hypomethylated sites (negative β values). Interestingly, despite hypomethylated sites, some genes exhibited hypermethylated sites (positive β values), such as ABCA7, BDNF, and WWOX. A 14-week CT regimen was adequate to induce differential methylation in 12 CE-related genes in healthy older women, alongside improvements in motor skills and blood pressure. In conclusion, this study suggest that combined training can be a strategy to improve physical fitness in older individuals, especially able to induce methylation alterations in genes sites related to development of AD. It is important to highlight that training should act as protective factor in older adults.

PAX1 and SOX1 Gene Methylation as a Detection and Triage Method for Cervical Intraepithelial Neoplasia Diagnosis.

Methylation assays have demonstrated potential as dependable and high-precision approaches for identifying or triaging individuals with cervical cancer (CA) or cervical intraepithelial neoplasia (CIN). Our investigation aimed to assess the efficacy of the diagnosis and triage of the PAX1/SOX1 methylation panel in detecting CIN or CA. A total of 461 patients with abnormal high-risk human papillomavirus (hrHPV) or cytology test results were recruited for this study. Each patient underwent an assortment of assessments, comprising a cytology test, hrHPV test, colposcopy examination, and PAX1 and SOX1 methylation tests. The extent of methylation of both genes demonstrates a positive correlation with the severity of CIN lesions and CA. To determine the correlation for patients with CIN2 or worse (CIN2+), the area under curve was 0.821 (95% CI: 0.782-0.853) for PAX1 and 0.800 (95% CI: 0.766-0.838) for SOX1, while for CIN3 or worse (CIN3+), 0.881 (95% CI: 0.839-0.908) for PAX1 and 0.867 (95% CI: 0.830-0.901) for SOX1. The PAX1/SOX1 methylation marker panel performed sensitivity and specificity of 77.16% and 91.67% for CIN2+, 84.76% and 90.50% for CIN3+, respectively. Regarding triaging hrHPV+ patients, the PAX1/SOX1 methylation test only referred 11.83% of the patients who are unnecessary for colonoscopy examination, which is comparatively lower than cytology, thereby signifying a promising triage strategy for hrHPV-positive women. Furthermore, we observed that the positive PAX1/SOX1 methylation test result for untreated CIN1 or fewer patients would result in a higher likelihood of progression upon a 24-month follow-up visit. The present investigation demonstrates that the PAX1/SOX1 methylation marker panel exhibits favorable diagnostic performance in CIN detection and holds the potential to be employed for individual CIN tests or hrHPV-positive triage.

Meta-analysis of the accuracy for RASSF1A methylation in bronchial aspirates for the diagnosis of lung cancer.

To establish the diagnostic accuracy of RASSF1A (Ras association domain family 1 isoform) methylation using bronchial aspirates as an auxiliary method for diagnosing lung cancer through a systematic review and meta-analysis. Studies published prior to October 30, 2022, were retrieved from the Embase, PubMed, Web of Science, and Wan Fang databases using the keywords "lung cancer", "RASSF1A", "methylation", and "bronchial aspirates". A fixed or random effect model was used to calculate the combined sensitivity, specificity, positive likelihood ratios (LR), negative LR, diagnostic odds ratio (DOR), along with the respective 95% confidence intervals (CIs) and the area under the curve (AUC) with Q index. The threshold effect was defined by using the Spearman correlation coefficient, and the Deeks funnel plot was generated to evaluate publication bias. Among the 12 trials that met the inclusion criteria, a total of 2388 participants were involved. The pooled results for the diagnosis of lung cancer were as follows, when compared to the pathological diagnosis: sensitivity of 0.47 (95% CI: 0.45-0.50), specificity of 0.96 (95% CI: 0.95-0.97), positive LR of 12.18 (95% CI: 8.96-16.55), negative LR of 0.56 (95% CI: 0.52-0.61), DOR of 24.05 (95% CI: 17.29-33.47), and AUC of 0.78 (Q index = 0.72), respectively. The sensitivity of the RASSF1A methylation assay was relatively low in a detailed subgroup analysis, fluctuating between 0.39 and 0.90, indicating a limitation in its diagnostic value for lung cancer. The RASSF1A methylation assay, on the other hand, demonstrated excellent specificity, suggesting a high exclusion value. Of note, the diagnostic sensitivity, specificity, DOR, and AUC for small cell lung cancer were 0.90 (0.84-0.94), 0.95 (0.94-0.97), 249.5 (103.94-598.8), and 0.98, respectively, showing that RASSF1A methylation was a promising biomarker for diagnosing small cell lung cancer with both high diagnostic and exclusion value. Furthermore, RASSF1A methylation using bronchial washings and bronchial aspirates showed a high AUC of 0.998 and 0.93, respectively, indicating excellent diagnostic performance. The methylation of RASSF1A in bronchial aspirates demonstrated a high level of diagnostic accuracy and has the potential to be a valuable supplementary diagnostic method, especially for identifying small cell lung cancer.

Performance of Human Gene EPB41L3 and HPV 16/18 Viral DNA Methylation to Triage hrHPV-Positive Women.

More evidence from population-based cohort studies is required to confirm the application of methylation-based biomarkers in real-world settings. The cross-sectional and 24-month cumulative triage performance of a novel methylation assay targeting the host gene EPB41LE and HPV16/18 DNA L1/L2 regions among hrHPV-positive women was evaluated based on a population-based cohort study from China. Overall methylation positivity was 12.4% among hrHPV-positive women. Methylation-positive women had significantly higher risks of hrHPV persistence at 12M and 24M follow-up (RR12M = 1.9, 95%CI: 1.5-2.6 and RR24M = 1.7, 95%CI: 1.2-2.5). For CIN2+, cross-sectional triage sensitivity of methylation was similar to HPV16/18 (70.6% vs. 64.7%, pexact = 1.000), but was lower than cytology (94.1%), although not significantly (pexact = 0.213). The specificity (91.2%) of methylation was significantly higher than other triage methods (p < 0.001 for all). The longitudinal sensitivity of methylation over 24M follow-up was 56.0%, lower (but not significantly so) than HPV16/18 (64.0%, pexact = 0.688) and cytology (76.0%, pexact = 0.125). Methylation testing showed high positive predictive values for CIN2+ (41.4% at baseline, 50.0% at 24-month), while the CIN2+ risk of methylation negative women (cNPV) remained considerable (2.5% at baseline, 6.9% at 24-month). Study findings indicate that methylation has better specificity and predictive values for the presence or development of cervical precancer and might therefore be considered for the strategy of HPV screening and methylation triage followed by immediate treatment of triage-positive women and delayed follow-up of hrHPV-positive/methylation-negative women.

Epigenetic modifications of 45S rDNA associates with the disruption of nucleolar organisation during Cd stress response in Pakchoi

The role of the nucleolus in Pakchoi response to Cd stress remains largely unknown. In this work, we focus on exploring the underling mechanism between nucleolus disruption and epigenetic modification in Cd stressed-Pakchoi. Our results indicated that the proportion of nucleolus disruption, decondensation of 45 S rDNA chromatin, and a simultaneous increase in 5' external transcribed spacer region (ETS) transcription were observed with increasing Cd concentration, accompanied by genome-wide alterations in the levels of histone acetylation and methylation. Further results showed that Cd treatment exhibited a significant increase in H3K9ac, H4K5ac, and H3K9me2 levels occurred in promoter regions of the 45 S rDNA. Additionally, DNA methylation assays in the 45 S rDNA promoter region revealed that individual site-specific hypomethylation may be engaged in the activation of 45 S rDNA transcription. Our study provides some molecular mechanisms for the linkage between Cd stress, rDNA epigenetic modifications, and nucleolus disintegration in plants.

Unraveling Transcriptome Profile, Epigenetic Dynamics, and Morphological Changes in Psoriasis-like Keratinocytes: "Insights into Similarity with Psoriatic Lesional Epidermis".

Keratinocytes are one of the primary cells affected by psoriasis inflammation. Our study aimed to delve deeper into their morphology, transcriptome, and epigenome changes in response to psoriasis-like inflammation. We created a novel cytokine mixture to mimic mild and severe psoriasis-like inflammatory conditions in cultured keratinocytes. Upon induction of inflammation, we observed that the keratinocytes exhibited a mesenchymal-like phenotype, further confirmed by increased VIM mRNA expression and results obtained from confocal microscopy. We performed RNA sequencing to achieve a more global view, revealing 858 and 6987 DEGs in mildly and severely inflamed keratinocytes, respectively. Surprisingly, we found that the transcriptome of mildly inflamed keratinocytes more closely mimicked that of the psoriatic epidermis transcriptome than the severely inflamed keratinocytes. Genes involved in the IL-17 pathway were a major contributor to the similarities of the transcriptomes between mildly inflamed KCs and psoriatic epidermis. Mild and severe inflammation led to the gene regulation of epigenetic modifiers such as HATs, HDACs, DNMTs, and TETs. Immunofluorescence staining revealed distinct 5-hmC patterns in inflamed versus control keratinocytes, and consistently low 5-mC intensity in both groups. However, the global DNA methylation assay detected a tendency of decreased 5-mC levels in inflamed keratinocytes versus controls. This study emphasizes how inflammation severity affects the transcriptomic similarity of keratinocytes to psoriatic epidermis and proves dynamic epigenetic regulation and adaptive morphological changes in inflamed keratinocytes.

Performance of SHOX2 and RASSF1A methylation assay in supernatants and matched cell pellets for the diagnosis of malignant pleural effusion

BackgroundIt is difficult to distinguish between malignant pleural effusion (MPE) and benign pleural effusion (BPE). The purpose of this study was to determine the best specimen type by evaluating the DNA methylation status of SHOX2 and RASSF1A in 3 matched PE components. MethodsIn total, 94 patients were enrolled, including 45 MPE, 35 BPE, and 14 undefined PE (UPE) with malignancies. PE samples were processed into supernatants, fresh-cell pellets, and formalin-fixed and paraffin-embedded (FFPE) cell blocks, respectively. A quantitative real-time PCR was used to detect the methylation status of SHOX2 and RASSF1A. ResultsSHOX2 and RASSF1A methylation levels were significantly higher in the 3 MPE sample types than those of BPE (P < 0.05). The area under the curve using cell-free DNA (cf-DNA) was the highest. The detection sensitivity of SHOX2 and RASSF1A in fresh-cell DNA, cf-DNA and FFPE cell-block were 71.1% (32/45), 97.8% (44/45) and 66.7% (28/42), respectively, with specificities of 97.1% (34/35), 94.3% (33/35), and 96.9% (31/32). Notably, a combination of the cytological analysis and cf-DNA methylation assay showed an increase in positivity rate from 75.6% to 100%. ConclusionsThe SHOX2 and RASSF1A methylation assay using cf-DNA, the primary recommended specimen type, can excellently increase the diagnostic sensitivity of MPE. A combination of methylation assay with cytological analysis can be used for auxiliary diagnosis of PE.

M6A‑modified HOXC10 promotes HNSCC progression via co‑activation of ADAM17/EGFR and Wnt/β‑catenin signaling.

The homeobox (HOX) gene family plays a fundamental role in carcinogenesis. However, the oncogenic mechanism of HOXC10 in head and neck squamous cell carcinoma (HNSCC) remains unclear. In the present study, it was revealed that HOXC10 expression was significantly higher in HNSCC tissues than in adjacent tissues, and a high level of HOXC10 was closely associated with worse clinical outcomes. HOXC10 overexpression promoted HNSCC cell proliferation, migration, and invasion, both invitro and invivo. Mechanistically, chromatin immunoprecipitation sequencing revealed that HOXC10 drove the transcriptional activation of a disintegrin and metalloproteinase17 (ADAM17), and the ADAM17/epidermal growth factor receptor (EGFR)/ERK1/2 signaling pathway facilitating the proliferation of HNSCC. Furthermore, mass spectrometric analysis indicated that HOXC10 interacted with ribosomal proteinS15A (RPS15A) and enhanced RPS15A protein expression, activating the Wnt/β‑catenin pathway and contributing to invasion and metastasis of HNSCC. Additionally, the methylated RNA immune precipitation and RNA antisense purification assays showed that N6‑methyladenosine (m6A) writer, methyltransferase‑like 3, catalyzed m6A modification of the HOXC10 transcript, m6A reader insulin like growth factor2 mRNA binding protein (IGF2BP)1 and IGF2BP3 involved in recognizing and stabilizing m6A‑tagged HOXC10 mRNA. In summary, the present study identified HOXC10 as a promising candidate oncogene in HNSCC. The m6A modification‑mediated HOXC10 promoted proliferation, migration, and invasion of HNSCC through co‑activation of ADAM17/EGFR and Wnt/β‑catenin signaling, providing a novel diagnostic and prognostic biomarker and a potential therapeutic target for HNSCC.

Challenges of recruitment and biomarkers collection in Caribbean Hispanics

AbstractBackgroundGenetic Studies of Alzheimer’s Disease in Caribbean Hispanics (CH) has been ongoing for over 20 years. Our primary goal was to identify genes in large well‐phenotyped, multiplex families, and sporadic cases and controls. This longitudinal collection of data represents a unique resource for the scientific community. The project is led by collaborators in the United States (US) and the Dominican Republic (DR). For the past three years, our efforts have been centered on the collection of cerebrospinal fluid (CSF), plasma, serum and Paxgene tubes for RNA to study core biomarkers. Collection of blood samples for DNA to conduct genetic analyses is also ongoing.MethodThe cohort consisted of participants aged 50 and older from the DR, including those living in the New York City area, willing and able to provide a blood and CSF samples. Interviewers in the DR were trained to collect data, blood samples and CSF. DNA samples were sent overnight to Columbia university (CU) for processing. CSF, Paxgene tubes, serum and plasma samples were processed in the DR and stored at ‐80F for batch shipping to CU. APOE genotyping was conducted in the US. Biomarkers, metabolomics, and proteomics data was generated at Columbia University. Methylation assays from Paxgene was conducted at Uniformed Services University (USUHS).ResultWe have databased a total of 1899 individuals with plasma samples, 1707 with Paxgene, 1731 with serum and 203 with CSF. Blood samples from 1079 participants have genome wide arra, whole genome sequencing and APOE genotyping. 192 CSF samples have proteomics completed, 153 CSF samples have metabolomics data and 742 have plasma biomarkers (Ab40, Ab42, T‐tau, P‐tau181, NFL and GFAP)).ConclusionSample collection for genetic studies in developing countries present important logistical challenges. Despite these challenges, sample quality is comparable to those collected in New York. Biomarkers from CSF, serum and plasma can give us a closer look into underrepresented populations such as Caribbean Hispanics. Results suggest that similar protocols can be successful in Latin America. Future work will augment the biosample collection of the cohort by collecting samples from additional subjects.

Biomarker collection from the Caribbean Hispanic families from Dominican Republic and New York.

AbstractBackgroundGenetic Studies of Alzheimer’s Disease in Caribbean Hispanics (CH) has been ongoing for over 20 years. Our primary goal was to identify genes in large well‐phenotyped, multiplex families, and sporadic cases and controls. This longitudinal collection of data represents a unique resource for the scientific community. The project is led by collaborators in the United States (US) and the Dominican Republic (DR). For the past three years, our efforts have been centered on the collection of cerebrospinal fluid (CSF), plasma, serum and Paxgene tubes for RNA to study core biomarkers. Collection of blood samples for DNA to conduct genetic analyses is also ongoing.MethodThe cohort consisted of participants aged 50 and older from the DR, including those living in the New York City area, willing and able to provide a blood and CSF samples. Interviewers in the DR were trained to collect data, blood samples and CSF. DNA samples were sent overnight to Columbia university (CU) for processing. CSF, Paxgene tubes, serum and plasma samples were processed in the DR and stored at ‐80F for batch shipping to CU. APOE genotyping was conducted in the US. Biomarkers, metabolomics, and proteomics data was generated at Columbia University. Methylation assays from Paxgene was conducted at Uniformed Services University (USUHS).ResultWe have databased a total of 1899 individuals with plasma samples, 1707 with Paxgene, 1731 with serum and 203 with CSF. Blood samples from 1079 participants have genome wide arra, whole genome sequencing and APOE genotyping. 192 CSF samples have proteomics completed, 153 CSF samples have metabolomics data and 742 have plasma biomarkers (Ab40, Ab42, T‐tau, P‐tau181, NFL and GFAP)).ConclusionSample collection for genetic studies in developing countries present important logistical challenges. Despite these challenges, sample quality is comparable to those collected in New York. Biomarkers from CSF, serum and plasma can give us a closer look into underrepresented populations such as Caribbean Hispanics. Results suggest that similar protocols can be successful in Latin America. Future work will augment the biosample collection of the cohort by collecting samples from additional subjects.

Abstract C058: Differential DNA methylation in HPV+ and HPV- penile tumors from Puerto Rican men

Abstract The incidence of penile squamous cell carcinoma (PSCC) in Puerto Rico is nearly three times higher when compared with other racial and ethnic groups in the United States. Mortality rate for PSCC is also significantly higher among Puerto Rican men when compared to the US population. Human papillomavirus (HPV) is a common virus, and its infection may increase the risk of developing PeCa. Despite the high incidence, the molecular mechanism of PSCC remains poorly understood. Our laboratory found that the prevalence of HPV in PSCC in Puerto Rican men is 49%. Due to the high rates of HPV infection and increased incidence of penile cancer in Puerto Rican men, there is a need to understand the pathogenesis of penile cancer. DNA Methylation is an epigenetic alteration that can be found in cancer cells, which can lead to changes in gene expression and cellular function. This study aims to analyze the DNA methylation patterns in HPV-positive and HPV-negative PSCC tissues from Puerto Rican men. DNA was extracted from twenty-four fresh PSCC tissue samples from Puerto Rican patients. HPV status and genotyping were determined using the DNA ELISA kit HPV SPF10 protocol. DNA was amplified and hybridized using PCR. We perform a DNA methylation assay using the Infinium Methylation EPIC v2.0 BeadChip Kit to identify and compare average DNA methylation levels (%) in the 9 HPV-positive and 15 HPV-negative penile cancer samples. Our results demonstrated 43 loci significantly different between HPV-positive and HPV-negative samples, with an adjusted p-value &amp;lt;0.1 (Benjamini-Hochberg method). Most of the hypermethylated loci were present in HPV-negative tumor samples. Additionally, we identified four methylated-differentially expressed genes: ANXA4, CACNA1C-AS1, UBXN2A, and DUOX1, which are found as uncharacterized proteins (open reading frames, ORFs) of the human genome with adjusted p-value &amp;lt;0.05. Studies have revealed that specific DNA methylation patterns could be linked with cancer development and progression. Identification of differentially methylated regions in HPV positive (HPV+) PSCC compared with HPV negative (HPV-) cases will provide much needed data to aid studies on early detection, prognosis, and development of therapeutic agents to reduce the morbidity and mortality from PeCa. Identification of differentially expressed genes and pathways in HPV positive (HPV+) PeCa compared with HPV negative (HPV-) cases will provide much needed data to aid studies on prevention, early detection, prognosis, and development of therapeutic agents to reduce the morbidity and mortality from PeCa. Citation Format: Yarelis M. Roque Reyes, María Sánchez-Vázquez, Josué Perez, Luis R. Llanos, Ingrid Montes, Jasmine Figueroa-Díaz, Carlos Rivera-Lopez, María Marcos, Antonio Puras, Magaly Martínez-Ferrer. Differential DNA methylation in HPV+ and HPV- penile tumors from Puerto Rican men [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 C058.

Associations of serum DNA methylation levels of chemokine signaling pathway genes with mild cognitive impairment (MCI) and Alzheimer's disease (AD).

To investigate the associations of serum DNA methylation levels of chemokine signaling pathway genes with Alzheimer's disease (AD) and mild cognitive impairment (MCI) in elderly people in Xinjiang, China, and to screen out genes whose DNA methylation could distinguish AD and MCI. 37 AD, 40 MCI and 80 controls were included in the present study. DNA methylation assay was done using quantitative methylation-specific polymerase chain reaction (qMSP). Genotyping was done using Sanger sequencing. DNA methylation levels of ADCY2, MAP2K1 and AKT1 were significantly different among AD, MCI and controls. In the comparisons of each two groups, AKT1 and MAP2K1's methylation was both significantly different between AD and MCI (p < 0.05), whereas MAP2K1's methylation was also significantly different between MCI and controls. Therefore, AKT1's methylation was considered as the candidate serum marker to distinguish AD from MCI, and its association with AD was independent of APOE ε4 allele (p < 0.05). AKT1 hypermethylation was an independent risk factor for AD and MAP2K1 hypomethylation was an independent risk factor for MCI in logistic regression analysis (p < 0.05). This study found that the serum of AKT1 hypermethylation is related to AD independently of APOE ε4, which was differentially expressed in the Entorhinal Cortex of the brain and was an independent risk factor for AD. It could be used as one of the candidate serum markers to distinguish AD and MCI. Serum of MAP2K1 hypomethylation is an independent risk factor for MCI.

Hypermethylated CDO1 and CELF4 in cytological specimens as triage strategy biomarkers in endometrial malignant lesions.

Developing a non-invasive and reliable triage test for endometrial malignant lesions is an important goal, as it could help to reduce the number of invasive diagnostic procedures required and improve patient survival. We aimed to estimate the diagnostic value of DNA methylation levels in cervical cytological samples of endometrial cancer (EC) and endometrial atypical hyperplasia (AH). A total of 607 women who had indications for endometrial biopsy in the Department of Obstetrics and Gynecology of Cangzhou Central Hospital from October 2022 to April 2023 were enrolled in this study. The cervical exfoliated cells were collected for gene methylation before endometrial biopsy. Clinical information, tumor biomarkers, and endometrial thickness (ET) of transvaginal ultrasonography (TVS) were also collected. With endometrial histopathology as the gold standard, multivariate unconditional logistic regression was applied to analyze the risk factors of endometrial malignant lesions. The role of cysteine dioxygenase type 1 (CDO1) and CUGBP Elav-like family member 4 (CELF4) gene methylation as a triage strategy biomarker in endometrial malignant lesions was specifically explored. Multivariate logistic regression analysis showed that premenopausal ET ≥ 11mm or postmenopausal ET ≥ 5mm, CDO1 ΔCt ≤ 8.4, or CELF4 ΔCt ≤ 8.8 were the risk factors for AH and EC, with odds ratios (ORs) (95%CI) of 5.03 (1.83-13.82) and 6.92 (1.10-43.44), respectively (p-values < 0.05). The sensitivity and specificity of CDO1/CELF4 dual-gene methylation assay for AH and EC reached 84.9% (95%CI: 75.3%-94.5%) and 86.6% (95%CI: 83.8%-89.5%), respectively. ET combined with DNA methylation detection further improved the specificity to (94.9%, 95%CI: 93.1%-96.8%). The accuracy of cervical cytology DNA methylation is superior to that of other clinical indicators in the non-invasive examination of endometrial malignant lesions. DNA methylation combined with TVS can further improve the specificity and is a promising biomarker triage strategy in women with suspected endometrial lesions.

Doxorubicin induced epigenetic regulation of dendritic cell maturation in association with T cell activation facilitates tumor protective immune response in non-small cell lung cancer (NSCLC)

BackgroundNSCLC is one of the leading causes of death and is often diagnosed at late stages with no alternative therapeutic approach. DCs are professional antigen-presenting cells and DC-based immunotherapy has been under the spotlight for its anti-cancer properties. Epigenetic modifications including DNA methylation and histone modification in DCs play a crucial role in regulating their functions such as maturation and activation,innate immune responses, T cell priming, antigen presentation, and cytokine production. In the current study, we investigated the anti-cancer properties of Doxorubicin at a noncytotoxic concentration that could be extrapolated as an epigenetic regulator for DC maturation to elicit anti-tumor activity. MethodologiesPBMCs from normal and NSCLC blood samples were isolated and treated with growth factors. DCs were matured with low dose Doxorubicin and the DC maturation markers were checked by using flow-cytometry. Further, ELISA was performed and low dose Doxorubicin-induced DCs were pulsed with LCA (Lung Cancer Antigen) and primed with CD4 +T helper (Th) cells for cytotoxicity assessment. Further, epigenetic markers of T: DC conjugation were immunofluorescently visualized under a microscope. ChIP-qPCR and Invitro assays such as histone methylation, DNA methylation, and m6A methylation were performed to study the epigenetic changes under low dose Dox treatment. IL-12 neutralization assay was performed to check for the IL-12 dependency of DCs and their effect under Dox at low dose treatment. This was further followed by a Western Blotting analysis for histone and non-histone proteins. ResultsLow dose Doxorubicin induces epigenetic changes in DCs to elicit an anti-tumor response in NSCLC through the generation of CTLs with a concomitant increase in the extracellular secretions of anti-inflammatory cytokines. We also found that low dosage of Doxorubicin matured DCs when pulsed with LCA and primed with CD4 +T helper cells, secrete IFN-γ which is important in orchestrating adaptive immunity by activating CD8 + cytotoxic T-lymphocytes. Also, the secretions of IL-12 help us infer that protective immunity is also induced via Th1 response which triggered selectively the translocation of PKCθ to immunological synapse in between DC and Th. Further, methylation and acetylation markers H3K4me3 and H3K14Ac respectively upregulated whereas levels of STAT5, NFkB, NOTCH1, and DNAPKcs were downregulated. DNA and RNA methylation assays then lead to confirmations about the epigenetic changes caused by low dose Dox treatment. DNA methylation was reduced which resulted in the activation of tumor suppressor gene p53 and Th1-associated transcription factor TBX21. On the other hand, both absolute and relative RNA methylation quantification increased in the presence of Dox at a low dose. ConclusionFrom this study, we understand that non-cytotoxic concentration of Doxorubicin increases the Ag-presenting ability of DCs via an IL-12-dependent mechanism and causes epigenetic modifications in NSCLC.

Trophectoderm biopsy of blastocysts following IVF and embryo culture increases epigenetic dysregulation in a mouse model.

Does trophectoderm biopsy (TEBx) of blastocysts for preimplantation genetic testing in the clinic affect normal placental and embryo development and offspring metabolic outcomes in a mouse model? TEBx impacts placental and embryonic health during early development, with some alterations resolving and others worsening later in development and triggering metabolic changes in adult offspring. Previous studies have not assessed the epigenetic and morphological impacts of TEBx either in human populations or in animal models. We employed a mouse model to identify the effects of TEBx during IVF. Three groups were assessed: naturally conceived (Naturals), IVF, and IVF + TEBx, at two developmental timepoints: embryonic day (E)12.5 (n = 40/Naturals, n = 36/IVF, and n = 36/IVF + TEBx) and E18.5 (n = 42/Naturals, n = 30/IVF, and n = 35/IVF + TEBx). Additionally, to mimic clinical practice, we assessed a fourth group: IVF + TEBx + Vitrification (Vit) at E12.5 (n = 29) that combines TEBx and vitrification. To assess the effect of TEBx in offspring health, we characterized a 12-week-old cohort (n = 24/Naturals, n = 25/IVF and n = 25/IVF + TEBx). Our mouse model used CF-1 females as egg donors and SJL/B6 males as sperm donors. IVF, TEBx, and vitrification were performed using standardized methods. Placenta morphology was evaluated by hematoxylin-eosin staining, in situ hybridization using Tpbpa as a junctional zone marker and immunohistochemistry using CD34 fetal endothelial cell markers. For molecular analysis of placentas and embryos, DNA methylation was analyzed using pyrosequencing, luminometric methylation assay, and chip array technology. Expression patterns were ascertained by RNA sequencing. Triglycerides, total cholesterol, high-, low-, and very low-density lipoprotein, insulin, and glucose were determined in the 12-week-old cohort using commercially available kits. We observed that at E12.5, IVF + TEBx had a worse outcome in terms of changes in DNA methylation and differential gene expression in placentas and whole embryos compared with IVF alone and compared with Naturals. These changes were reflected in alterations in placental morphology and blood vessel density. At E18.5, early molecular changes in fetuses were maintained or exacerbated. With respect to placentas, the molecular and morphological changes, although different compared to Naturals, were equivalent to the IVF group, except for changes in blood vessel density, which persisted. Of note is that most differences were sex specific. We conclude that TEBx has more detrimental effects in mid-gestation placental and embryonic tissues, with alterations in embryonic tissues persisting or worsening in later developmental stages compared to IVF alone, and the addition of vitrification after TEBx results in more pronounced and potentially detrimental epigenetic effects: these changes are significantly different compared to Naturals. Finally, we observed that 12-week IVF + TEBx offspring, regardless of sex, showed higher glucose, insulin, triglycerides, lower total cholesterol, and lower high-density lipoprotein compared to IVF and Naturals, with only males having higher body weight compared to IVF and Naturals. Our findings in a mouse model additionally support the need for more studies to assess the impact of new procedures in ART to ensure healthy pregnancies and offspring outcomes. Data reported in this work have been deposited in the NCBI Gene Expression Omnibus under accession number GSE225318. This study was performed using a mouse model that mimics many clinical IVF procedures and outcomes observed in humans, where studies on early embryos are not possible. This study highlights the importance of assaying new procedures used in ART to assess their impact on placenta and embryo development, and offspring metabolic outcomes. This work was funded by a National Centers for Translational Research in Reproduction and Infertility grant P50 HD068157-06A1 (M.S.B., C.C., M.M.), Ruth L. Kirschstein National Service Award Individual Postdoctoral Fellowship F32 HD107914 (E.A.R.-C.) and F32 HD089623 (L.A.V.), and National Institutes of Health Training program in Cell and Molecular Biology T32 GM007229 (C.N.H.). No conflict of interest.

Integrated genetic and epigenetic analyses uncovered GLP1R association with metabolically healthy obesity.

Both genetic and epigenetic variations of GLP1R influence the development and progression of obesity. However, the underlying mechanism remains elusive. This study aims to explore the mediation roles of obesity-related methylation sites in GLP1R gene variants-obesity association. A total of 300 Chinese adult participants were included in this study and classified into two groups: 180 metabolically healthy obesity (MHO) cases and 120 metabolically healthy normal-weight (MHNW) controls. Questionnaire investigation, physical measurement and laboratory examination were assessed in all participants. 18 single nucleotide polymorphisms (SNPs) and 31 CpG sites were selected for genotype and methylation assays. Causal inference test (CIT) was performed to evaluate the associations between GLP1R genetic variation, DNA methylation and MHO. The study found that rs4714211 polymorphism of GLP1R gene was significantly associated with MHO. Additionally, methylation sites in the intronic region of GLP1R (GLP1R-68-CpG 7.8.9; GLP1R-68-CpG 12.13; GLP1R-68-CpG 17; GLP1R-68-CpG 21) were associated with MHO, and two of these methylation sites (GLP1R-68-CpG 7.8.9; GLP1R-68-CpG 17) partially mediated the association between genotypes and MHO. Not only the gene polymorphism, but also the DNA methylation of GLP1R was associated with MHO. Epigenetic changes in the methylome may in part explain the relationship between genetic variants and MHO.

NIMG-57. PERFORMANCE OF DEEP LEARNING IN MGMT PROMOTER METHYLATION STATUS PREDICTION USING BRAIN MRI: RESULTS FROM A LARGE COHORT OF IDH-WILDTYPE GLIOMAS TESTED BY A SINGLE METHYLATION ASSAY

Abstract There have been several attempts to predict the MGMT promoter methylation status of gliomas using deep learning (DL), with mixed results. These studies did not account for IDH mutation status and did not utilize a uniform methylation assay, introducing potential confounders in the experimental design. We assessed the performance of DL applied to brain MRI to predict the MGMT promoter methylation status of IDH-wildtype gliomas tested with a standardized assay. We included 539 IDH-wildtype gliomas tested with real-time methylation-specific PCR from 2017-2022; 205 (38%) were MGMT promoter methylated. The assay targets 8 downstream MGMT CpG sites using ACTB as control; methylation status is determined based on the difference of PCR crossing-point values: methylated if difference &amp;lt; 5, unmethylated if difference &amp;gt; 7, indeterminate otherwise. We used post-contrast T1 and T2 MRIs as inputs for our models trained using stratified 5-fold cross-validation, 400 epochs, batch size of 16, a learning rate scheduler starting at 10-3, weighted binary cross-entropy loss, and AdamW optimizer. We repeated the experiments using two 3D DL architectures (DenseNet121 and EfficientNetB0) and two image preprocessing pipelines: the BraTS protocol and one that uses padding instead of registration to an MRI atlas, keeping constant other experimental variables. For Densenet121, the best cross-validation AUROCC and F1-score (median, median absolute deviation) were 0.67 (0.06), 0.59 (0.02) using the BraTS protocol, and 0.67 (0.06), 0.58 (0.01) using padding. For EfficientNetB0, were 0.67 (0.02), 0.66 (0.01) using the BraTS protocol and 0.67 (0.01), 0.68 (0.01) using padding. Our findings suggest that it is unlikely that DL models trained on brain MRI can predict the MGMT promoter methylation of IDH-wildtype gliomas. Importantly, our study is the first to focus on an IDH-wildtype adult diffuse glioma cohort tested with a single, standardized MGMT methylation assay, eliminating the variability introduced by different assays and IDH status.

EPCO-40. CARM1 IS A NOVEL TUMOR SUPPRESSOR IN GLIOBLASTOMA THAT REGULATES QKI THROUGH ARGININE METHYLATION

Abstract Glioblastoma (GBM) is the most common form of primary brain tumor, characterized by dismal patient prognosis. Current pharmacological interventions have not significantly improved patient outcomes due to intratumor heterogeneity and the persistence of glioma stem cells (GSCs) that drive invasion and resistance. Thus, treatment strategies based on a deeper molecular understanding of gliomagenesis are urgently needed. Quaking (Qki, encoded by the gene Qk) is frequently altered in GBM; Qk mutations or deletions occur in 35% percent of cases, and the Qki protein is absent in 50-60% of cases. Our lab found that deletion of Qk in Trp53-/-; Pten-/- pre-malignant neural stem cells (NSCs) enables them to expand outside their niche and develop into malignant GBM. Mechanistically, we determined that Qki functions as a transcriptional coactivator of lipid metabolism genes in the central nervous system, where loss of Qki dysregulates the lipidome and alters cellular membrane integrity. While several downstream processes governed by Qki have been delineated, upstream regulators of Qki remain unknown. Recurrent cancer mutations to specific Qki arginine residues implicate the dysregulation of arginine methylation, and previous studies indicate that proteins in the same family as Qki can be methylated by protein arginine methyltransferases (PRMTs). Utilizing in vitro methylation assays, we demonstrate that Qki is selectively methylated by the PRMT coactivator-associated methyltransferase 1 (CARM1) in the Quaking regulatory domain (QRD), where cancer mutations were reported. Within the QRD, we identified arginine 242 (R242) and arginine 256 (R256) as specific CARM1-mediated methylation sites on Qki. Mutations to these sites compromise Qki transcriptional activity, as Qki methylation mutant-expressing GSCs exhibited downregulation of Qki target genes compared to those expressing wildtype Qki. Taken together, we provide evidence that CARM1 functions as a tumor suppressor in GBM through a novel mechanism of Qki dysregulation.

A sensitive and robust plasma-based DNA methylation panel for early detection of target gastrointestinal cancers

BackgroundTarget gastrointestinal cancers (GICs), encompassing esophageal cancer (EC), gastric cancer (GC), and colorectal cancer (CRC), originate within a single readily accessible luminal organ system and are diagnosable using endoscopy. However, endoscopy is an invasive procedure with low compliance and no plasma-based DNA methylation assay for the early detection of GICs. MethodsNine potential DNA methylation markers were identified and evaluated in tissue (n=60) and plasma (n=155) cohorts to select the most suitable markers. A training cohort (n=244) and a validation cohort (n=199), including GICs patients, benign tumors, gastrointestinal polyps, and controls, were enrolled to develop and validate a DNA methylation panel. An independent prospective cohort (n=158) was used to validate the panel's performance and compare it with blood protein tumor markers. ResultsSix out of nine candidate methylation markers with excellent discrimination abilities in both tissue and plasma cohorts were selected for the DNA methylation panel. The panel demonstrated high AUC values of 0.937 (EC), 0.968 (GC), and 0.987 (CRC) in training cohort, and achieved AUC values of 0.921 (EC), 0.921 (GC), and 0.959 (CRC) in validation cohort. Notably, it achieved impressive AUC values of 0.971 and 0.843 for identifying stage I GICs in the training and validation cohorts, respectively. In the prospective cohort, the six-marker panel showed comparable AUC values to CEA, AFP, and CA19-9 (0.935, 0.769, 0.663, and 0.668, respectively). ConclusionThis study successfully developed and validated a novel, robust, sensitive, and specific plasma-based DNA methylation panel, offering a promising strategy for the early detection of GICs.