Methylation Status Of Gene Promoter Research Articles

Optimizing Droplet Digital PCR Assay for Precise Assessment of MEIS1 Gene Promoter Methylation

Abstract DNA methylation is characterized as a gene regulatory mechanism that involves the methylation of the 5-carbon (C5) position of cytosine, resulting in the formation of 5-methylcytosine. The analysis of aberrantly methylated cytosine-phosphate-guanine (CpG) dinucleotides, primarily in the promoter regions of tumor suppressor genes, can serve as promising prognostic and predictive markers of cancer development. Meis homeobox 1 (MEIS1) gene, crucial for cell growth and differentiation, exhibits dysregulation linked to various cancer types, acting as both a positive and negative regulator. The selection of an appropriate method for the evaluation of gene promoter methylation status is important for clinical implementation without biases regarding false positive and false negative outcomes. The study focuses on the optimization of a novel droplet digital PCR (ddPCR) assay for identifying the methylation status of MEIS1. Compared to traditional methods, ddPCR offers an increased sensitivity and specificity, presenting a promising tool for precise DNA methylation assessment with potential implications for cancer diagnostics and prognostics.

Abstract 7395: Histology-based prognosis prediction using deep learning outperforms and is independent of the MGMT methylation status in patients with glioblastoma

Abstract Introduction: MGMT gene promoter methylation status is today the only biomarker of survival for GBM patients and predicts favorable response to temozolomide (TMZ). Here we propose a deep learning-based model using histology data (H&E slides) to predict the prognosis of patients with Glioblastoma (GBM) treated by standard of care (SOC). Methods: We included TCGA GBM patients and selected only patients treated after 2005 (Stupp protocol start); compliant to WHO 2021 classification; treated by surgery and adjuvant therapy; with a follow-up superior to median overall survival (OS) (14.5 months). Short and long survivor labels were defined based on median OS cut-off. A deep learning pipeline was trained to predict these labels from the H&E slides (baseline surgery samples). First, the slides were automatically segmented into tissue and background, divided into a regular grid of tiles (112 × 112µm), and tiles were vectorized using a vision transformer-based feature extractor trained on H&E slides of 5,500 patients with 16 cancer subtypes (GBM excluded). In the second step, ABMIL deep learning model was trained to predict the survival category using the bag of tile features available from step 1 for each patient. The cohort was divided into five folds with even numbers of short and long survivors. For each fold, 50 ABMIL models were trained on the remaining folds and ensembled to predict the category of the patients in the fold. Training hyperparameters were tuned on a separated cohort excluding GBM patients. Results: After filtering, we analyzed n = 192 TCGA GBM patients including 154 patients with a known MGMT promoter methylation status. Our histology-based model had an accuracy of 0.661 and a ROC AUC of 0.706. It stratified the population into short and long survivors with an Hazard Ratio (HR) of 0.48 (95% CI 0.34-0.69). Our model outperformed the MGMT methylation status that had an HR of 0.57 (95% CI 0.40-0.83). Moreover, our model was independent of MGMT methylation status and the combination of both was able to further improve prognosis prediction: we applied our model independently to the 91 patients with an unmethylated MGMT status (predicted: 43 low, 48 high) and to the 63 patients with a methylated MGMT status (predicted: 30 low, 33 high), and obtained HR of 0.42 (95% CI 0.27-0.67) and 0.49 (95% CI 0.28-0.86) respectively. Conclusion: Having selected patients treated by the latest SOC and classified as GBM by the latest WHO criteria, we propose a model predicting survival based on histological specimens available in routine care, which outperformed the current unique biomarker MGMT methylation status. The combination of this status and our model improves prognosis predictions compared to each strategy alone. Citation Format: Lucas Fidon, Celine Thiriez, Alexandre Grimaldi, Omar Darwiche Domingues, Charles Maussion, Caroline Hoffmann. Histology-based prognosis prediction using deep learning outperforms and is independent of the MGMT methylation status in patients with glioblastoma [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 7395.

Expression and Promoter Methylation Status of CDX1 and CDX2 Genes in Chinese Patients with Gastric Cancer

Expression and Promoter Methylation Status of CDX1 and CDX2 Genes in Chinese Patients with Gastric Cancer

DNA Methylation of the IL-17A Gene Promoter Is Associated with Subclinical Atherosclerosis and Coronary Artery Disease: The Genetics of Atherosclerotic Disease Mexican Study.

The interleukin-17 (IL-17) has a crucial role during inflammation and has been associated with cardiovascular diseases, but its role in epigenetics is still poorly understood. Therefore, the aim of this study was to evaluate the DNA methylation status of the IL-17A gene promoter to establish whether it may represent a risk factor for subclinical atherosclerosis (SA) or clinical coronary artery disease (CAD). We included 38 patients with premature CAD (pCAD), 48 individuals with SA, and 43 healthy controls. Methylation in the CpG region of the IL-17A gene promoter was assessed via methylation-specific polymerase chain reaction (MSP). Individuals with SA showed increased methylation levels compared to healthy controls and pCAD patients, with p < 0.001 for both. Logistic regression analysis showed that high methylation levels represent a significant risk for SA (OR = 5.68, 95% CI = 2.38-14.03, p < 0.001). Moreover, low methylation levels of the IL-17A gene promoter DNA represent a risk for symptomatic pCAD when compared with SA patients (OR = 0.16, 95% CI = 0.06-0.41, p < 0.001). Our data suggest that the increased DNA methylation of the IL-17A gene promoter is a risk factor for SA but may be a protection factor for progression from SA to symptomatic CAD.

Targeting the ‘DNA methylation mark’: Analysis of early epigenetic-alterations in children chronically exposed to arsenic

Chronic exposure to arsenic causes adverse health effects in children. Aberrant epigenetic modifications including altered DNA methylation pattern are one of the major steps towards malignant transformation of cells. Our group has previously identified significant alteration in DNA methylation mark in arsenic exposed adults, affecting major biological pathways. Till date, no information is available exploring the altered DNA methylation mark in telomere regulation and altered mitochondrial functionality in association with DNA damage in arsenic-exposed children. Our study aims in identifying signature epigenetic pattern associated with telomere lengthening, mitochondrial functionality and DNA damage repair in children with special emphasis on DNA methylation. Biological samples (blood and urine) and drinking water were collected from the children aged between 5 and 16 years of arsenic exposed areas (N = 52) of Murshidabad district and unexposed areas (N = 50) of East Midnapur districts, West Bengal, India. Methylation-specific PCR was performed to analyse subtelomeric methylation status and promoter methylation status of target genes. Results revealed altered DNA methylation profile in the exposed children compared to unexposed. Promoter hypermethylation was observed in MLH1 and MSH2 (p < 0.05 and p < 0.001) indicating inefficiency in DNA damage repair. Hypomethylation in mitochondrial D-loop (p < 0.05) and TFAM promoter region (p < 0.05) along with increased mitochondrial DNA copy number among exposed children was also observed. Significant increase in telomere length and region specific subtelomeric hypermethylation (XpYp, p < 0.05) was found. Analysis of S-Adenosyl Methionine (SAM) and 8-oxoDG level revealed significant depletion of SAM (p < 0.001) and elevated oxidative DNA damage (p < 0.001) respectively in arsenic toxicity. Our study identified key methylation patterns in arsenic-exposed children which may act as an early predictive biomarker in the near future. Further in-depth studies involving large sample size and transcriptomic analysis are required for understanding the mechanistic details.

Role of epigenetics and the transcription factor Sp1 in the expression of the D prostanoid receptor 1 in human cartilage

D prostanoid receptor 1 (DP1), a prostaglandin D2 receptor, plays a central role in the modulation of inflammation and cartilage metabolism. We have previously shown that activation of DP1 signaling downregulated catabolic responses in cultured chondrocytes and was protective in mouse osteoarthritis (OA). However, the mechanisms underlying its transcriptional regulation in cartilage remained poorly understood. In the present study, we aimed to characterize the human DP1 promoter and the role of DNA methylation in DP1 expression in chondrocytes. In addition, we analyzed the expression level and methylation status of the DP1 gene promoter in normal and OA cartilage. Deletion and site-directed mutagenesis analyses identified a minimal promoter region (−250/−120) containing three binding sites for specificity protein 1 (Sp1). Binding of Sp1 to the DP1 promoter was confirmed using electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assays. Treatment with the Sp1 inhibitor mithramycin A reduced DP1 promoter activity and DP1 mRNA expression. Inhibition of DNA methylation by 5-Aza-2′-deoxycytidine upregulated DP1 expression, and in vitro methylation reduced the DP1 promoter activity. Neither the methylation status of the DP1 promoter nor the DP1 expression level were different between normal and OA cartilage. In conclusion, our results suggest that the transcription factor Sp1 and DNA methylation are important determinants of DP1 transcription regulation. They also suggest that the methylation status and expression level of DP1 are not altered in OA cartilage. These findings will improve our understanding of the regulatory mechanisms of DP1 transcription and may facilitate the development of intervention strategies involving DP1.

Methylation Patterns of Fetal Hemoglobin Regulating Genes in High-Risk (HR) -MDS Patients Following Treatment with Azacytidine

Background: Current standard of care for HR-MDS patients, i.e. hypomethylating agent (HMAs) treatment with azacytidine (AZA) or decitabine (DAC), is associated with a 30-50% response rate, and many efforts have been made to identify predictive factors for response. Fetal hemoglobin (HbF) levels have been proposed as a prognostic index for DAC treatment (Stomper J. et al, 2019), but no study has examined its prognostic value in AZA treated patients, and the mechanism of γ-globin chain re-expression has not been investigated. Methods: Bone marrow (BM) aspirates of 28 HR-MDS or CMML patients were collected at baseline and at the first evaluation of response, following 6 cycles of AZA treatment. Methylation of Cytosine followed by Guanosine (CpG) sites of the γ-globin gene promoter and CpG 326 of ZBTB7A/LRF, a previously characterized epigenetic repressor of HbF (Chondrou V. et al, 2022), were detected with pyrosequencing analysis in total genomic DNA, extracted from BM samples, pre- and post-treatment. Response to AZA was characterized as marrow response (mCR), when blasts were &amp;lt;5% and were reduced &amp;gt;50% from baseline, and as hematological response (HI-R) when there was at least one lineage improvement. HbF was measured with HPLC from the peripheral blood of 20 patients, at 5 time-points (before 0, 1, 2, 4 and 6-7 cycles of treatment), to test its expression pattern and potential prognostic significance. HbF values &amp;gt;1% were considered induced, as in previous studies (Stomper J. et al, 2019). For statistical analysis IBM SPSS 28.0.0.0 program was used. One patient with extremely high HbF before treatment (HbF 0=13.4%) was excluded from the analysis, as well as the CpG site 3 of γ-globin gene promoter, which was 100% methylated pre- and post- AZA treatment. Results: Median baseline HbF levels were 0.6% (range 0 - 2.6), and they were increased to 0.8% (range 0 - 1.8) and to 1.1% (range 0 - 3.5), at 2 and 6 months, post-AZA treatment, respectively. Difference in the repeated measurements of HbF was of borderline statistical significance (p=0.068), specified between cycles 2 and 6 (p=0.038). Correlation analysis showed a significant association of mCR with induced HbF after 6 cycles of treatment (p=0.011, r s=0.618) and a strong positive correlation between elevation of HbF at cycle 2 and time to leukemia progression (p=0.019, r s=0.886). Overall, median methylation of the γ-globin gene promoter was 73.4% and 72.8% pre- and post-AZA treatment, respectively (p=0.159). However, patients exhibiting any kind of response showed reduced methylation at site 2 post- AZA, at the limit of statistical significance (p=0.095 for mCR patients and p=0.066 for HI-R), whereas site 4 was significantly hypermethylated among HI-R patients (p=0.042). A negative correlation between methylation of the γ-globin gene promoter and gene expression was also identified (p=0.008, r s=-0.427), particularly at the sites 2 and 5 (p 2=0.011, p 5=0.004). Methylation analysis of CpG 326 of ZBTB7A/LRF showed significant hypermethylation at site 1 (p=0.044) and reduction of methylation at site 22 (p=0.043), following AZA treatment. When analysis was restricted among responders only, there was significant hypermethylation at site 1 (p 1=0.020) and borderline significant hypermethylation at sites 5 and 11, among mCR patients (p 5=0.069, p 11=0.079). Conversely, sites 9 and 13 were significantly hypermethylated among non-responders (p 9=0.034, p 13=0.047). With Spearman's analysis, a significant negative correlation between methylation fold change in site 13 and mCR was observed (p=0.002, r s=-0.555). Although no correlation between HbF and CpG 326 methylation status pre- treatment was observed, after 6 cycles of AZA a borderline correlation was identified between HbF levels and fold change of mean methylation of CpG 326 (p=0.05, r s=0.482). Moreover, induced HbF levels were related at the limit of statistical significance to increase in methylation at sites 1,3,5,11 and 22 (p 1=0.077, p 3=0.052, p 5=0.087, p 11=0.061, p 22=0.060). Conclusions: In this study, a relationship of γ-globin gene promoter methylation status and HbF levels was found among HR-MDS patients, treated with AZA. Moreover, hypermethylation in CpG326 of ZBTB7A/LRF after AZA treatment was associated with HbF levels, and different methylation patterns were observed between responders and non-responders. Further research is needed to clarify the role of LRF in MDS response to AZA.

Dysregulation of DNA epigenetic modulators during prostate carcinogenesis in an eastern Indian patient population: Prognostic implications

The role of epigenetic alteration in prostate cancer pathogenesis was reported. We aimed to analyze dysregulation of DNA methylase (DNA methyl transferase/DNMT) and demethylase (ten eleven translocase/TET) and the associated interplay between them during prostate tumorigenesis. Promoter methylation and RNA/protein expression of selected DNMT and TETs were analysed in normal prostate, benign prostatic hyperplasia (BPH), and prostate cancer (PCa). Genomic 5-hydroxymethylcytosine (5hmC) level was detected and correlated with DNMT and TET proteins. Clinicopathological association of molecular data was done. Our data revealed a very low frequency of promoter methylation for DNMT1 (5-3% and high frequency for TET1 (22–38%), TET2 (68–90 %), and TET3 (43-32 %) in BPH and PCa. The promoter methylation of DNMT1 (p = 0.019) showed a significantly decreasing trend, while that of TET1 (p = 0.0005) and TET2 (p < 0.0001) showed an increasing trend from normal prostate to BPH to PCa, indicating their epigenetic dysregulation during prostate tumorigenesis. RNA/protein overexpression of DNMT1 and reduced expression of TET1 and TET2 in PCa compared to BPH were associated with the promoter methylation status of genes. The 5hmC level was significantly lower in PCa than in BPH and correlated negatively with DNMT1 but positively with TET1 and TET2 proteins, suggesting dysregulation of DNA methylase and de-methylase activities during prostate tumorigenesis. Lastly, tumors having methylated TET1 and TET2 promoters showed advanced clinicopathological features (a higher PSA level/Gleason score) and increased risk of bone metastasis. In conclusion, DNMT1 upregulation and epigenetic silencing of TET1 and TET2 was seen during PCa development. TET1 and TET2 promoter methylation has prognostic importance.

Comprehensive assessment of mismatch repair and microsatellite instability status in molecular classification of endometrial carcinoma

Objective: To explore the concordance and causes of different mismatch repair (MMR) and microsatellite instability (MSI) detection results in endometrial carcinoma (EC) molecular typing. Methods: A total of 214 EC patients diagnosed from January 2021 to April 2023 were selected at the Department of Pathology, Peking University Third Hospital. The immunohistochemistry (IHC) results of MMR protein were reviewed. Tumor specific somatic mutations, MMR germline mutations, microsatellite scores and tumor mutation burden (TMB) were detected by next-generation sequencing (NGS) with multi-gene panel. Methylation-specific PCR was used to detect the methylation status of MLH1 gene promoter in cases with deficient MLH1 protein expression. In cases with discrepant results between MMR-IHC and MSI-NGS, the MSI status was detected again by PCR (MSI-PCR), and the molecular typing was determined by combining the results of TMB and MLH1 gene promoter methylation. Results: (1) In this study, there were 22 cases of POLE gene mutation subtype, 55 cases of mismatch repair deficient (MMR-d) subtype, 29 cases of p53 abnormal subtype, and 108 cases of no specific molecular profile (NSMP). The median age at diagnosis of MMR-d subtype (54 years old) and the proportion of aggressive histological types (40.0%, 22/55) were higher than those of NSMP subtype [50 years old and 12.0% (13/108) respectively; all P<0.05]. (2) Among 214 patients, MMR-IHC test showed that 153 patients were mismatch repair proficient (MMR-p), 49 patients were MMR-d, and 12 patients were difficult to evaluate directly. MSI-NGS showed that 164 patients were microsatellite stable (MSS; equal to MMR-p), 48 patients were high microsatellite instability (MSI-H; equal to MMR-d), and 2 patients had no MSI-NGS results because the effective sequencing depth did not meet the quality control. The overall concordance between MMR-IHC and MSI-NGS was 94.3% (200/212). All the 12 discrepant cases were MMR-d or subclonal loss of MMR protein by IHC, but MSS by NGS. Among them, 10 cases were loss or subclonal loss of MLH1 and (or) PMS2 protein. Three discrepant cases were classified as POLE gene mutation subtype. In the remaining 9 cases, 5 cases and 3 cases were confirmed as MSI-H and low microsatellite instability (MSI-L) respectively by MSI-PCR, 6 cases were detected as MLH1 gene promoter methylation and 7 cases demonstrated high TMB (>10 mutations/Mb). These 9 cases were classified as MMR-d EC. (3) Lynch syndrome was diagnosed in 27.3% (15/55) of all 55 MMR-d EC cases, and the TMB of EC with MSH2 and (or) MSH6 protein loss or associated with Lynch syndrome [(71.0±26.2) and (71.5±20.1) mutations/Mb respectively] were significantly higher than those of EC with MLH1 and (or) PMS2 loss or sporadic MMR-d EC [(38.2±19.1) and (41.9±24.3) mutations/Mb respectively, all P<0.01]. The top 10 most frequently mutated genes in MMR-d EC were PTEN (85.5%, 47/55), ARID1A (80.0%, 44/55), PIK3CA (69.1%, 38/55), KMT2B (60.0%, 33/55), CTCF (45.5%, 25/55), RNF43 (40.0%, 22/55), KRAS (36.4%, 20/55), CREBBP (34.5%, 19/55), LRP1B (32.7%, 18/55) and BRCA2 (32.7%, 18/55). Concurrent PTEN, ARID1A and PIK3CA gene mutations were found in 50.9% (28/55) of MMR-d EC patients. Conclusions: The concordance of MMR-IHC and MSI-NGS in EC is relatively high.The discordance in a few MMR-d EC are mostly found in cases with MLH1 and (or) PMS2 protein loss or MMR protein subclonal staining caused by MLH1 gene promoter hypermethylation. In order to provide accurate molecular typing for EC patients, MLH1 gene methylation, MSI-PCR, MMR gene germline mutation and TMB should be combined to comprehensively evaluate MMR and MSI status.

Identification of ALX3 Gene Promoter Hypermethylation as a Potential Biomarker for Lung Cancer.

Recent studies imply the significance of promoter CpG methylation as a biomarker for various cancer types in different genes. ALX3 is one of the candidate genes with prominent promoter methylation status change. In this study, the methylation status of ALX3 gene promoter and its expression was analyzed in lung cancer cell lines and clinical samples from The Cancer Genome Atlas Program (TCGA) program. Methylation status was screened using the COBRA Assay, and gene expression in two cancer (A549 & H1299) and one normal (Beas 2B) lung cell lines was determined using RT-PCR. ALX3 gene promoter was found to be hypermethylated in both lung cancer cell lines compared to normal cells. However, no difference in the expression of this gene was observed. In addition to our in vitro findings, DNA Methylation and RNA-seq data of 413 adenocarcinoma samples from TCGA-LUAD dataset were analyzed. ALX3 gene was found to be hypermethylated in tumor compared to normal samples. Interestingly, the expression level of ALX3 gene in tumors was found to be higher than that in normal samples. ALX3 gene promoter hypermethylation could serve as a biomarker in lung cancer.

Mutational profiling of newly diagnosed glioblastoma to identify predictive biomarkers of efficacy and safety of anlotinib combination therapy.

e14037 Background: Glioblastoma (GBM) is highly vascular tumor with the specific pathologic feature of microvascular proliferation. Our previous phase Ⅱ clinical trial (NCT04119674) has firstly demonstrated the antiangiogenic multikinase inhibitor anlotinib combined with STUPP regimen can lengthen progression-free survival (PFS) in patients with newly diagnosed GBM. However, no biomarker for angiogenic therapy has been proved yet. Therefore, the second analysis was performed to identify the predictive biomarkers. Methods: A total of 21 patients with newly diagnosed GBM were enrolled in the retrospective research. The methylation status of the MGMT gene promoter was examined using the MDxHealth PCR assay. DNA-based whole genome sequencing was undertaken to detect the genomic alterations of patients. Results: The differences of gene aberrances between patients who reached and those who did not reach the primary endpoint were observed. ATRX and MYO15A mutations were only found in patients who had yet not experienced relapse. On contrast, the mutations of PIK3CA, HEG1, HMCN1 and RP1L1 occurred only in the recurrent group. HEG1 (HR 6.0, 95% CI 1.2-28.8, p= 0.026) and RP1L1 (HR 12.5, 95% CI 2.1-74.3, p= 0.005), genes related to angiogenesis, were both independent biomarkers of PFS. RP1L1 was also significantly associated with shorter overall survival (OS) (HR 33.4, 95% CI 3.2-351.4, p= 0.003). Interestingly, PIK3CA alterations were significantly higher in patients with cerebral than in those without ( p= 0.032, OR 16.4). Conclusions: Our study suggested that alterations in HEG1, RP1L1 and PIK3CA could be novel biomarkers for the newly diagnosed GBM patients received anti-angiogenesis combination therapy, and provided now insights for the precise treatment of GBM.

The relationship of promoter methylation of calcium voltage-gated channel alpha 1 and interleukin-16 to primary osteoarthritis.

Osteoarthritis is the most prevalent joint disease worldwide and the primary cause of musculoskeletal dysfunction. Epigenetic changes in various genes, particularly methylation, have been implicated as possible underlying causes of primary osteoarthritis. The aim of our study was to investigate the promoter methylation status of the calcium voltage-gated channel alpha 1 subunit G (CACNA1G) and interleukin-16 (IL-16) genes, which are strongly associated with calcium channel activity and antigen presentation, respectively, in primary osteoarthritis patients. Twenty-one patients with primary osteoarthritis and 25 healthy controls were included in our study. The methylation status of CACNA1G and IL-16 genes was analyzed with methylation-specific Polymerase Chain Reaction (PCR), and the serum levels of IL-16 were determined with Enzyme-Linked Immunosorbent Assay (ELISA). The age of the patients was 63.95±8.41 years, and they were 15 females and 6 males. The promoter of the CACNA1G gene was found to be hypermethylated in primary osteoarthritis patients (p<0.001). In contrast, the promoter of the IL-16 gene was found to be hypomethylated compared to the control (p<0.001). The serum levels of IL-16 increased in parallel with the hypomethylated promoter status of IL-16 gene in primary osteoarthritis patients compared to the control (p<0.001). Our study indicates that the methylation status of CACNA1G and IL-16 gene promoters are epigenetically altered in patients with primary osteoarthritis. Moreover, increased serum IL-16 levels in osteoarthritis patients may be associated with hypomethylation of the IL-16 gene promoter.

458 Assessment of MGMT Methylation Status Using High-Performance Liquid Chromatography in Newly Diagnosed Glioblastoma

INTRODUCTION: The utility of O6-methylguanine-DNA methyltransferase (MGMT) gene promoter methylation status as a prognostic marker in patients with glioblastoma (GBM) has been established. However, the number of CpG sites that must be methylated to cause transcriptional silencing remains unclear, and no significant consensus exists on the optimal method of assessing MGMT methylation. METHODS: W analyzed the MGMT methylation status of 28 isocitrate dehydrogenase–wild-type GBMs treated with temozolomide using ion-exchange HPLC and bisulfite sequencing using frozen samples. We designed three primers for separate regions (regions 1–3) that had 21 to 38 CpGs for PCR and validated the MGMT promoter methylation status. RESULTS: There was a strong correlation between HPLC and bisulfite sequencing results (R = 0.794). The optimal cutoff values for MGMT methylation in HPLC were determined to allow differentiation of patient prognosis by receiver operating characteristic curve analysis. The cutoff values were 34.15% for region 1, 8.84% for region 2, and 36.72% for region 3. Kaplan–Meyer curve analysis estimated that the most differentiated prognosis was enabled in the setting of 8.84% methylation of MGMT in region 2. Progression-free survival and overall survival were significantly longer for patients in this setting of region 2 methylation (p = 0.00365 and p = 0.00258, respectively). CONCLUSIONS: The combination of our HPLC method and the original primer setting provides a new standard method for determination of MGMT methylation status in patients with GBM and is useful for refining MGMT-based drug selection.

Adipose Tissue-Mesenchymal Stem Cells Caused to Change the Methylation Status of hTERT Gene Promoter CpG Islands of Molt-4 Leukemia Cells as Cell-based Therapy.

DNA methylation was considered as prognostic information in some hematological malignancies. Previous studies have reported the in vitro and in vivo biology role of mesenchymal stem cells (MSCs) on leukemic cells. The aim of this study was to investigate the effect of MSCs on the promoter methylation status of hTERT as a catalytic subunit of telomerase enzyme. In the experimental study, the Molt-4 leukemic cells were co-cultured with MSCs for 7 days. At the end of the co-culture period, the Molt-4 cells were collected, DNA and protein were extracted. Then methylation specific-PCR and western blotting were done for evaluating the hTERT gene promoter methylation status and cyclin D1 and hTERT protein expression, respectively. In the following, the flow cytometry was done for cell cycle distribution assay. It was found that MSCs resulted in a significant decrease in the cyclin D1 and hTERT protein expression levels. Also, MSCs caused changes in the methylation status of the CpG islands in the hTERT gene promoter region. The following results showed that MSCs caused a significant increase in the number of cells at G0/G1 phase and arrest the G0/G1 phase as well as decrease in the cell proliferation of Molt-4 cells. It is concluded that co-culture of MSCs with Molt-4 cells could be involved in changing the methylation status of hTERT gene promoter, cell cycle and hTERT protein expression; it could be potentially beneficial for further investigations regarding the cell transplantation and cell-based therapy.

Evaluation of BRCA1 Gene Promoter Methylation Status in Sporadic Breast Cancer Patients in Southwest of Iran.

Similar to other types of cancer, the development of breast cancer is a multi-stage process, consisting of various mutations and epigenetic changes in many genes. Mutations in the BRCA1 gene, which is a tumor suppressor gene, are considered as the most important types of mutations. The pivotal role of epigenetics is currently considered as the primary key to carcinogenesis. Several studies have previously reported the BRCA1 epigenetic silencing through promoter methylation in the pathophysiology of breast cancer cells. This study aimed to investigate whether the BRCA1 gene promoter methylation in peripheral blood cells is correlated with the risk of breast cancer. In the current study, DNA samples were extracted from blood cells belonged to 74 patients with breast cancer as well as 30 healthy individuals, and the BRCA1 gene promoter methylation status in these two groups was examined using Methylation Specific PCR (MSP). out of 74 patients, 2 cases demonstrated methylation in their BRCA1 gene promoter; however, none of the healthy controls demonstrated methylation status. Among these 74 patients, 13 cases were at the early stages (stage I), and two patients who had BRCA1 gene methylation (15.4%), were in this group (p=0.02). While 34 and 27 patients were at stages II and III, respectively, showing a negative state of BRCA1 gene methylation. Although 2 out of 74 patients resulted positive for methylation status, the healthy controls demonstrated no methylation. Consequently, there was inadequate evidence to confirm the association between BRCA1 gene promoter methylation in blood and the risk of developing breast cancer.

PCDH17 gene promoter methylation status in a cohort of Egyptian women with epithelial ovarian cancer

Background and objectiveOvarian cancer is a leading cause of female mortality. Epigenetic changes occur in early stages of carcinogenesis and represent a marker for cancer diagnosis. Protocadherin 17 (PCDH17) is a tumor suppressor gene involved in cell adhesion and apoptosis. The methylation of PCDH17 gene promoter has been described in several cancers including ovarian cancer. The aim of the study was to compare the methylation status of PCDH17 gene promoter between females diagnosed with epithelial ovarian cancer and a control group composed of normal and benign ovarian lesions.MethodsFifty female subjects were included in our study (25 ovarian cancer patients and 25 controls). DNA was extracted from Formalin-Fixed Paraffin-Embedded (FFPE) tissues of the subjects. Methylation levels for six CpG sites in the PCDH17 gene promoter were assessed by pyrosequencing.ResultsThe methylation levels at five out of six sites were significantly higher in females with epithelial ovarian cancer compared to the control group. Moreover, the same applies for the mean methylation level with p value 0.018.ConclusionMethylation of PCDH17 gene promoter plays a role in ovarian carcinogenesis and can be used for diagnosis and early detection.

Association between multiple gene promoter hypermethylation and the risk of gastric cancer: A systematic review and meta-analysis.

Several studies have demonstrated an association between multiple gene hypermethylation and gastric cancer. However, the intrinsic mechanisms remain elusive and highly debatable. To this end, our study aims to investigate the correlation between the methylation status of multiple gene promoters and gastric cancer. PubMed, EMBASE, CNKI, WanFang, Cqvip, and Cochrane Library were queried from inception to May 2021, and the relationship between the methylation status of the CpG islands and gastric cancer risk was systematically assessed under the inclusion and exclusion criteria. The incidence of DNA methylation between tumor and non-tumor tissues was compared, and the clinicopathological significance of DNA methylation in gastric carcinoma was further evaluated. The odds ratio (OR) was estimated with a 95% confidence interval (CI), and forest plots were generated using the fixed-effects or random-effects model. In total, 201 studies were enrolled, and a higher frequency of CpG islands methylation was identified in gastric cancer tissues than in non-neoplastic tissues. This suggests that aberrant polygene methylation might be associated with the initial onset and progression of gastric cancer. This study sheds light on the significance of polygene methylation status in gastric cancer. The DNA methylation of these genes may serve as underlying epigenetic biomarkers, providing a promising molecular diagnostic approach for human gastric cancer clinical diagnosis. More large randomized trials are needed to confirm the findings.

Maternal exercise upregulates the DNA methylation of Agtr1a to enhance vascular function in offspring of hypertensive rats.

The angiotensin II signaling system regulates vascular dysfunction and is involved in the programming of hypertension. Maternal exercise has been linked to both short-term and long-term benefits for the mother and fetus. However, the impacts of maternal exercise on the intravascular renin-angiotensin system (RAS) in hypertensive offspring remain unexamined. This study examined whether maternal exercise has an epigenetic effect in repressing angiotensin II type 1 receptor (AT1R) expression, which leads to favorable alterations in the mesenteric artery (MA) function of spontaneously hypertensive offspring. Spontaneously hypertensive rats (SHRs) and Wistar-Kyoto (WKY) pregnant rats were randomly divided into an exercise group and a control group. Blood pressure, vascular tone, AT1R protein and mRNA expression, and AT1R gene (Agtr1a) promoter methylation status were examined in the MAs of 3-month-old male offspring. Maternal exercise significantly reduced the resting blood pressure and cardiovascular reactivity of offspring from SHRs. Furthermore, Ang II-AT1R activity in regulating vascular tone and AT1R expression was decreased in the MAs of the SHR offspring from the exercise groups. Importantly, exercise during gestation suppressed AT1R expression via hypermethylation of the Agtr1a promoter region and upregulated DNA methyltransferase (DNMT) expression in MAs of SHR offspring. These results suggest that maternal exercise upregulates DNMT expression, resulting in hypermethylation and repression of the Agtr1a gene, which may prevent MA dysfunction in the offspring of SHRs. A mechanistic model on the epigenetics of exercise during pregnancy. Maternal exercise during pregnancy triggers hypermethylation and transcriptional suppression of the Agtr1a gene via increased DNMT1 and DNMT3B expression in MAs of SHR offspring. Downregulation of AT1R expression reduces the contribution of Ang II to vascular tone, ultimately improving vascular structure and function. VSMC vascular smooth muscle cell; Ang II angiotensin II; AT1aR angiotensin type 1 receptor (AT1R) alpha subtypes; Agtr1a AT1R alpha isoform gene; MAs mesenteric arteries; BP blood pressure.

Targeted alpha therapy for glioblastoma.

According to the 2021 World Health Organization Classification of Tumors of the Central Nervous System, glioblastoma (GB) is a primary brain tumor and presents with the worst prognosis. Due to its infiltrating characteristic, molecular heterogeneity, and only partly preserved function of the blood-brain barrier, the median overall survival time is short (9-15 months), regardless of comprehensive treatment including surgery, radiotherapy, and chemotherapy. Several novel treatment strategies are under investigation. Unfortunately, none of them produced successful results; 90% of patients have a recurrence of the disease within 6 months. Local administration of the drug could be a promising approach to delivering treatment with minimized side effects, due to the recurrence of 95% glioblastomas in a margin of 2 cm at the primary site. Several ligand-receptor systems have been evaluated, such as targeting tenascin, the extracellular matrix protein, or radiolabeled somatostatin analogs, as it is overexpressed with the SSTR-2 receptor system in around 80% of gliomas. Moreover, this study revealed that the NK-1 receptor is overexpressed in GB, suggesting that substance P (SP) may serve as a ligand. A variety of radioisotopes, beta- (131I, 90Y, or 177 Lu) and alpha emitters (213Bi, 225Ac, or 211At), with different physical properties were tested for treatment. Alpha particles have many advantages over beta radiation such as short range with higher linear energy transfer. According to that characteristic, it is extremely dose delivered to the targeted cells, while reducing harm to nearby healthy tissue. Additionally, the biological effect of alpha radiation is independent of the cell cycle phase, cell oxygenation and O-6-methylguanine-DNA methyltransferase (MGMT) gene promoter methylation status. In this article, we summarize the experience with local treatment of primary and secondary GBs with locally used radioisotopes such as [213Bi]Bi-DOTA-SP or [225Ac]Ac-DOTA-SP.

Deep DNA sequencing of MGMT, TP53 and AGT in Mexican astrocytoma patients identifies an excess of genetic variants in women and a predictive biomarker.

Astrocytomas are a type of malignant brain tumor with an unfavorable clinical course. The impact of AGT and MGMT somatic variants in the prognosis of astrocytoma is unknown, and it is controversial for TP53. Moreover, there is a lack of knowledge regarding the molecular characteristics of astrocytomas in Mexican patients. We studied 48 Mexican patients, men and women, with astrocytoma (discovery cohort). We performed DNA deep sequencing in tumorsamples, targeting AGT, MGMT and TP53, and we studied MGMT gene promoter methylation status. Then we compared our findings to a cohort which included data from patients with astrocytoma from The Cancer Genome Atlas (validation cohort). In the discovery cohort, we found a higher number of somatic variants in AGT and MGMT than in the validation cohort (10.4% vs < 1%, p < 0.001), and, in both cohorts, we observed only women carried variants AGT variants. We also found that the presence of either MGMT variant or promoter methylation was associated to better survival and response to chemotherapy, and, in conjunction with TP53 variants, to progression-free survival. The occurrence of AGT variants only in women expands our knowledge about the molecular differences in astrocytoma between men and women. The increased prevalence of AGT and MGMT variants in the discovery cohort also points towards possible distinctions in the molecular landscape of astrocytoma among populations. Our findings warrant further study.