Genome-Wide Methylation Patterns in Primary Uveal Melanoma: Development of MethylSig-UM, an Epigenomic Prognostic Signature to Improve Patient Stratification.

Despite studies highlighting the prognostic utility of DNA methylation in primary uveal melanoma (pUM), it has not been translated into a clinically useful tool. We sought to define a methylation signature to identify newly diagnosed individuals at high risk for developing metastasis. Methylation profiling was performed on 41 patients with pUM with stage T2-T4 and at least three years of follow-up using the Illumina Infinium HumanMethylation450K BeadChip (N = 24) and the EPIC BeadChip (N = 17). Findings were validated in the TCGA cohort with known metastatic outcome (N = 69). Differentially methylated probes were identified in patients who developed metastasis. Unsupervised consensus clustering revealed three epigenomic subtypes associated with metastasis. To identify a prognostic signature, recursive feature elimination and random forest models were utilized within repeated cross-validation iterations. The 250 most commonly selected probes comprised the final signature, named MethylSig-UM. MethylSig-UM could distinguish individuals with pUM at diagnosis who develop future metastasis with an area under the curve of ~81% in the independent validation cohort, and remained significant in Cox proportional hazard models when combined with clinical features and established genomic biomarkers. Altered expression of immune-modulating genes were detected in MethylSig-UM positive tumors, providing clues for pUM resistance to immunotherapy. The MethylSig-UM model is available to enable additional validation in larger cohort sizes including T1 tumors.

Analysis of PD-L1 promoter methylation combined with immunogenic context in pancreatic ductal adenocarcinoma.

Despite the successful application of programmed cell death ligand 1 (PD-L1)-blocking strategies in some types of cancers and well-established prognostic indicators in pancreatic ductal adenocarcinoma (PDAC), the biological and clinical implications of the methylation status of PD-L1/PD-L2 in PDAC remain largely unknown. Therefore, this study aimed to explore the biological role of PD-L1/PD-L2 methylation and its association with clinicopathological features, clinical outcomes, and the immune microenvironment by analyzing the data on PD-L1/PD-L2 methylation and mRNA expression in PDAC cohorts obtained from the Cancer Genome Atlas and International Cancer Genome Consortium. The correlation between PD-L1 promoter methylation and PD-L1 expression and survival was further validated in an independent validation cohort (Peking Union Medical College Hospital [PUMCH] cohort) using pyrosequencing and immunohistochemistry. These results demonstrated that hypomethylation of the PD-L1 promoter was strongly associated with upregulated PD-L1 expression and shorter overall survival in PDAC. Multivariate Cox regression analyses revealed that the PD-L1 promoter methylation was an independent prognostic factor. PD-L1 promoter hypomethylation and high expression were related to aggressive clinical phenotypes. Moreover, both PD-L1 and PD-L2 methylation correlated with immune cell infiltration and the expression of immune checkpoint genes. PD-L1 promoter methylation status was further validated as an independent prognostic biomarker in patients with PDAC using the PUMCH cohort. The prognostic significance of PD-L1 promoter methylation was more discriminative in tumors with perineural/lymphovascular invasion and distant metastasis than in those without perineural/lymphovascular invasion and distant metastasis. In summary, the methylation status of the PD-L1 promoter is a promising biomarker for survival outcomes, immune infiltration, and the potential immune benefits of immunotherapy in PDAC.

A Common OXTR Risk Variant Alters Regulation of Gene Expression by DNA Hydroxymethylation in Pregnant Human Myometrium.

Postpartum hemorrhage, or excessive bleeding after birth, is a leading cause of maternal morbidity. A major cause of postpartum hemorrhage is uterine atony, tiring of the uterus which leads to ineffective contractions. Uterine contractions depend on oxytocin signaling in the myometrium, which in turn depends on expression of the oxytocin receptor (OXTR). Both genetic and epigenetic factors related to the oxytocin receptor are associated with risk of postpartum hemorrhage, but a mechanism relating these factors to oxytocin receptor activity in myometrium remains unclear. We report a genetic by epigenetic interaction whereby the relationship between DNA hydroxymethylation and OXTR gene expression depends on a common OXTR gene variant (rs53576). We also provide evidence that a similar genetic by epigenetic interaction using blood-derived DNA methylation is associated with relevant clinical outcomes: quantity of oxytocin administration and odds for postpartum hemorrhage. These results provide new avenues for predicting how women will respond to pharmacological agents in the prevention and treatment of postpartum hemorrhage.

Potential biomarkers: The hypomethylation of cg18949415 and cg22193385 sites in colon adenocarcinoma.

Overall cancer hypomethylation had been identified in the past, but it is not clear exactly which hypomethylation site is the more important for the occurrence of cancer. To identify key hypomethylation sites, we studied the effect of hypomethylation in twelve regions on gene expression in colon adenocarcinoma (COAD). The key DNA methylation sites of cg18949415, cg22193385 and important genes of C6orf223, KRT7 were found by constructing a prognostic model, survival analysis and random combination prediction a series of in-depth systematic calculations and analyses, and the results were validated by GEO database, immune microenvironment, drug and functional enrichment analysis. Based on the expression values of C6orf223, KRT7 genes and the DNA methylation values of cg18949415, cg22193385 sites, the least diversity increment algorithm were used to predict COAD and normal sample. The 100 % reliability and 97.12 % correctness of predicting tumor samples were obtained in jackknife test. Moreover, we found that C6orf223 gene, cg18949415 site play a more important role than KRT7 gene, cg22193385 site in COAD. In addition, we investigate the impact of key methylation sites on three-dimensional chromatin structure. Our results will be help for experimental studies and may be an epigenetic biomarker for COAD.

The Transcription Factor HOXA5: Novel Insights into Metabolic Diseases and Adipose Tissue Dysfunction.

The transcription factor HOXA5, from the HOX gene family, has long been studied due to its critical role in physiological activities in normal cells, such as organ development and body patterning, and pathological activities in cancer cells. Nonetheless, recent evidence supports the hypothesis of a role for HOXA5 in metabolic diseases, particularly in obesity and type 2 diabetes (T2D). In line with the current opinion that adipocyte and adipose tissue (AT) dysfunction belong to the group of primary defects in obesity, linking this condition to an increased risk of insulin resistance (IR) and T2D, the HOXA5 gene has been shown to regulate adipocyte function and AT remodeling both in humans and mice. Epigenetics adds complexity to HOXA5 gene regulation in metabolic diseases. Indeed, epigenetic mechanisms, specifically DNA methylation, influence the dynamic HOXA5 expression profile. In human AT, the DNA methylation profile at the HOXA5 gene is associated with hypertrophic obesity and an increased risk of developing T2D. Thus, an inappropriate HOXA5 gene expression may be a mechanism causing or maintaining an impaired AT function in obesity and potentially linking obesity to its associated disorders. In this review, we integrate the current evidence about the involvement of HOXA5 in regulating AT function, as well as its association with the pathogenesis of obesity and T2D. We also summarize the current knowledge on the role of DNA methylation in controlling HOXA5 expression. Moreover, considering the susceptibility of epigenetic changes to reversal through targeted interventions, we discuss the potential therapeutic value of targeting HOXA5 DNA methylation changes in the treatment of metabolic diseases.

Circulating NPTX2 methylation as a non-invasive biomarker for prognosis and monitoring of metastatic pancreatic cancer.

BACKGROUND: Pancreatic cancer is the most lethal cancer with a dismal prognosis mainly due to diagnosis at advanced stage and ineffective treatments. CA19-9 levels and computed tomography (CT) imaging are the main standard criteria for evaluating disease progression and treatment response. In this study we explored liquid biopsy-based epigenetic biomarkers for prognosis and monitoring disease in patients with metastatic pancreatic ductal adenocarcinoma (mPDAC). METHODS: Plasma samples were collected from 44 mPDAC patients at the time of diagnosis, and in 15 of them, additional samples were obtained during follow-up of the disease. After cell-free DNA (cfDNA), isolation circulating levels of methylated NPTX2, SPARC, BMP3, SFRP1 and TFPI2 genes were measured using digital droplet PCR (ddPCR). BEAMing technique was performed for quantitation of RAS mutations in cfDNA, and CA19-9 was measured using standard techniques. RESULTS: NPTX2 was the most highly and frequently methylated gene in cfDNA samples from mPDAC patients. Higher circulating NPTX2 methylation levels at diagnosis were associated with poor prognosis and efficiently stratified patients for prediction of overall survival (6.06% cut-off, p = 0.0067). Dynamics of circulating NPTX2 methylation levels correlated with disease progression and response to therapy and predicted better than CA19-9 the evolution of disease in mPDAC patients. Remarkably, in many cases the disease progression detected by CT scan was anticipated by an increase in circulating NPTX2 methylation levels. CONCLUSIONS: Our study supports circulating NPTX2 methylation levels as a promising liquid biopsy-based clinical tool for non-invasive prognosis, monitoring disease evolution and response to treatment in mPDAC patients.

Chemo-Enzymatic Fluorescence Labeling Of Genomic DNA For Simultaneous Detection Of Global 5-Methylcytosine And 5-Hydroxymethylcytosine.

5-Methylcytosine and 5-hydroxymethylcytosine are epigenetic modifications involved in gene regulation and cancer. We present a new, simple, and high-throughput platform for multi-color epigenetic analysis. The novelty of our approach is the ability to multiplex methylation and de-methylation signals in the same assay. We utilize an engineered methyltransferase enzyme that recognizes and labels all unmodified CpG sites with a fluorescent cofactor. In combination with the already established labeling of the de-methylation mark 5-hydroxymethylcytosine via enzymatic glycosylation, we obtained a robust platform for simultaneous epigenetic analysis of these marks. We assessed the global epigenetic levels in multiple samples of colorectal cancer and observed a 3.5-fold reduction in 5hmC levels but no change in DNA methylation levels between sick and healthy individuals. We also measured epigenetic modifications in chronic lymphocytic leukemia and observed a decrease in both modification levels (5-hydroxymethylcytosine: whole blood 30 %; peripheral blood mononuclear cells (PBMCs) 40 %. 5-methylcytosine: whole blood 53 %; PBMCs 48 %). Our findings propose using a simple blood test as a viable method for analysis, simplifying sample handling in diagnostics. Importantly, our results highlight the assay's potential for epigenetic evaluation of clinical samples, benefiting research and patient management.

Development and validation of a simple general population lung cancer risk model including AHRR-methylation.

INTRODUCTION: Screening reduces lung cancer mortality of high-risk populations. Currently proposed screening eligibility criteria only identify half of those individuals, who later develop lung cancer. This study aimed to develop and validate a sensitive and simple model for predicting 10-year lung cancer risk. METHODS: Using the 1991-94 examination of The Copenhagen City Heart Study in Denmark, 6,820 former or current smokers from the general population were followed for lung cancer within 10 years after examination. Logistic regression of baseline variables (age, sex, education, chronic obstructive pulmonary disease, family history of lung cancer, smoking status and cumulative smoking, secondhand smoking, occupational exposures to dust and fume, body mass index, lung function, plasma C-reactive protein, and AHRR(cg05575921) methylation) identified the best predictive model. The model was validated among 3,740 former or current smokers from the 2001-03 examination, also followed for 10 years. A simple risk chart was developed with Poisson regression. RESULTS: Age, sex, education, smoking status, cumulative smoking, and AHRR(cg05575921) methylation identified 65 of 88 individuals who developed lung cancer in the validation cohort. The highest risk group, consisting of less educated men aged >65 with current smoking status and cumulative smoking >20 pack-years, had absolute 10-year risks varying from 4% to 16% by AHRR(cg05575921) methylation. CONCLUSION: A simple risk chart including age, sex, education, smoking status, cumulative smoking, and AHRR(cg05575921) methylation, identifies individuals with 10-year lung cancer risk from below 1% to 16%. Including AHRR(cg05575921) methylation in the eligibility criteria for screening identifies smokers who would benefit the most from screening.

The prognostic value and immune correlation of IL18 expression and promoter methylation in renal cell carcinoma.

BACKGROUND: Renal cell carcinoma (RCC) is not sensitive to immunotherapy and has poor prognosis. DNA methylation regulates gene expression, and its abnormal changes are related to many human diseases. Recently, DNA methylation has been found to participate in immune infiltration in various cancers. However, its pattern in RCC remains poorly understood. RESULTS: We found that IL18 was significantly over-expressed in RCC tumor tissues compared to normal adjacent tissues The IL18 promoter region was hypomethylated, which was strongly correlated with elevated IL18 mRNA expression, and predicted advanced clinicopathological characteristics and shorter overall survival. Furthermore, we found that IL18 promoter methylation was significantly related to the down-regulation of immune checkpoint molecules and increase of CD8 + T cell infiltration in RCC tumor tissues. CONCLUSIONS: We have identified the important role of IL18 promoter methylation and expression, which are associated with clinicopathological characteristics, overall survival, immune cell infiltration and expression of immune checkpoint molecules in RCC. We present the rationale for IL18 promoter methylation as a molecular biomarker for predicting the response of RCC to immune checkpoint inhibitors.

The Relevance of DNA Methylation and Histone Modification in Periodontitis: A Scoping Review.

Background: Periodontitis is a chronic inflammatory disease involving an interplay between bacteria, inflammation, host response genes, and environmental factors. The manifestation of epigenetic factors during periodontitis pathogenesis and periodontal inflammation is still not well understood, with limited reviews on histone modification with periodontitis management. This scoping review aims to evaluate current evidence of global and specific DNA methylation and histone modification in periodontitis and discuss the gaps and implications for future research and clinical practice. Methods: A scoping literature search of three electronic databases was performed in SCOPUS, MEDLINE (PubMed) and EMBASE. As epigenetics in periodontitis is an emerging research field, a scoping review was conducted to identify the extent of studies available and describe the overall context and applicability of these results. Results: Overall, 30 studies were evaluated, and the findings confirmed that epigenetic changes in periodontitis comprise specific modifications to DNA methylation patterns and histone proteins modification, which can either dampen or promote the inflammatory response to bacterial challenge. Conclusions: The plasticity of epigenetic modifications has implications for the future development of targeted epi-drugs and diagnostic tools in periodontitis. Such advances could be invaluable for the early detection and monitoring of susceptible individuals.

Role of DNA De-methylation intermediate '5-hydroxymethylcytosine' in ovarian cancer management: A comprehensive review.

Ovarian cancer remains the most eminent silent killer, with high morbidity and mortality among all gynaecological cancers. The advanced-stage patient's diagnosis has a low survival rate caused by its asymptomatic progression and diverse histopathological sub-types, wherefore in poor prognosis and highly recurring malignancy with multidrug resistance towards chemotherapy. Epigenetic biomarkers open promising avenues of intriguing research to combat OC malignancy, furthermore a tool for its early diagnosis. 5-hydroxymethycytosine (5-hmC), alias the sixth base of the genome, is an intermediate formed during the recently established DNA demethylation process and catalysed via ten-eleven translocation (TET) family of enzymes. It plays a significant role in regulating gene expression and has sparked interest in various cancer types. This review summarizes the role of active DNA demethylation process, its enzymes and intermediate 5-hmC in epigenetic landscape of ovarian cancer as a potent biomarker for clinical translation in identification of therapeutic targets, diagnostic and prognostic evaluation.

Prognostic value of JAK3 promoter methylation and mRNA expression in clear cell renal cell carcinoma.

INTRODUCTION: Janus kinase 3 (JAK3) is a well-established oncogene in clear cell renal cell carcinoma (ccRCC). The methylation status of oncogene promoters has emerged as biomarkers for cancer diagnosis and prognosis. OBJECTIVE: This study aims to investigate the biological and clinical significance of JAK3 promoter methylation in ccRCC. METHODS: We analyzed the relationship of JAK3 promoter methylation with its mRNA expression, overall survival, and immune cell infiltration in a cohort obtained from The Cancer Genome Atlas (TCGA), which was further validated by another independent cohort. We further validated correlations of JAK3 promoter methylation with JAK3 expression, overall survival, and immune cell infiltration in an independent ccRCC cohort (Sun Yat-sen University Cancer Center (SYSUCC) cohort) by methods of immunohistochemistry (IHC) and pyrosequencing. RESULTS: We found JAK3 promoter was significantly hypomethylated in tumor tissues compared to normal adjacent tissues in ccRCC, and JAK3 promoter hypomethylation was strongly correlated with high JAK3 mRNA expression in all three ccRCC cohorts we examined. JAK3 promoter hypomethylation predicted advanced clinicopathological characteristics and shorter overall survival (TCGA cohort and SYSUCC cohort). Furthermore, we found that JAK3 promoter methylation was significantly associated with immune cell infiltration and expression of immune checkpoint molecules (TCGA cohort and CPTAC cohort). Finally, our SYSUCC cohort validated that JAK3 promoter methylation was correlated with CD4+ and CD8+ T cell infiltration in ccRCC tumor tissues. CONCLUSION: Our data demonstrated that the crucial role of JAK3 promoter methylation in its expression regulation and tumor microenvironment. JAK3 promoter methylation and expression are associated with clinicopathological characteristics, overall survival, and immune cell infiltration in ccRCC. We propose a rationale for further validation of JAK3 promoter methylation as a molecular biomarker for predicting responses to immune checkpoint inhibitors in ccRCC.

SEPTIN9-SDC2-VIM methylation signature as a biomarker for the early diagnosis of colorectal cancer.

The accurate detection of colorectal cancer (CRC) at its initial stage can reduce mortality. However, the broad application of endoscopy has been limited due to the invasive procedure and patient noncompliance. Liquid biopsy with subsequent mapping of methylation in specific cell-free DNA (cfDNA) may represent an alternative approach for early diagnosis. In this study, we have developed a minimal-invasive blood-based test for detection of precancerous lesions and early-stage CRC. Using TCGA M450K methylation data, we identified candidate methylation sites with the highest Fold Change (FC) for three genes (SEPTIN9, SDC2 and VIM), which were selected from previous studies. Based on logistic regression models, we developed a 3-gene methylation signature for CRC diagnosis with high accuracy (Sensitivity =0.959, Specificity =1, AUC =0.997). Using independent public databases and data from blood samples, this model has demonstrated superior performance. The AUC was 0.919-1 and 0.905-0.916 in public tissue database for CRC and blood sample data, respectively. Thus, our proposed 3-gene methylation signature has a more reliable performance than other methods. Furthermore, signal enhancement effect of 3-gene methylation signature can improve the accuracy of early diagnosis for CRC, which demonstrates the potential for clinical application.

MCC Gene Silencing Is a CpG Island Methylator Phenotype-Associated Factor That Predisposes Colon Cancer Cells to Irinotecan and Olaparib.

Chemotherapy is a mainstay of colorectal cancer treatment, and often involves a combination drug regime. CpG island methylator phenotype (CIMP)-positive tumors are potentially more responsive to the topoisomerase-inhibitor irinotecan. The mechanistic basis of the increased sensitivity of CIMP cancers to irinotecan is poorly understood. Mutated in Colorectal Cancer (MCC) is emerging as a multifunctional tumor suppressor gene in colorectal and liver cancers, and has been implicated in drug responsiveness. Here, we found that CIMP tumors undergo MCC loss almost exclusively via promoter hypermethylation rather than copy number variation or mutations. A subset of cancers display hypomethylation which is also associated with low MCC expression, particularly in rectal cancer, where CIMP is rare. MCC knockdown or deletion was found to sensitize cells to SN38 (the active metabolite of irinotecan) or the PARP-inhibitor Olaparib. A synergistic effect on cell death was evident when these drugs were used concurrently. The improved SN38/irinotecan efficacy was accompanied by the down-regulation of DNA repair genes. Thus, differential methylation of MCC is potentially a valuable biomarker to identify colorectal cancers suitable for irinotecan therapy, possibly in combination with PARP inhibitors.

Evaluation of a Prognostic Epigenetic Classification System in Chronic Lymphocytic Leukemia Patients.

BACKGROUND: Methylation at 5 CpG sites was previously shown to classify chronic lymphocytic leukemia (CLL) into 3 prognostic subgroups. Here, we aimed to validate the marker set in an additional cohort and to evaluate its clinical utility for CLL patient stratification. METHODS: We evaluated this epigenetic marker set in 79 German patients using bisulfite treatment followed by pyrosequencing and classification using a support vector machine-learning tool. RESULTS: The n-CLL, i-CLL, and m-CLL classification was detected in 28 (35%), 10 (13%), and 41 (51%) patients, respectively. Epigenetic grouping was associated with IGHV mutational status (P = 2 × 10-12), isolated del13q (P = 9 × 10-6), del17p (P = .015), complex karyotype (P = .005), VH-usage, and clinical outcome as time to first treatment (P = 1.4 × 10-12) and overall survival (P = .003). Multivariate Cox regression analysis identified n-CLL as a factor for earlier treatment hazard ratio (HR), 6.3 (95% confidence interval [CI] 2.4-16.4; P = .0002) compared to IGHV mutational status (HR 4.6, 95% CI 1.9-11.3, P = .0008). In addition, when comparing the prognostic value of the epigenetic classification system with the IGHV classification, epigenetic grouping performed better compared to IGHV mutational status using Kaplan-Meier estimation and allowed the identification of a third, intermediate (i-CLL) group. Thus, our study confirmed the prognostic value of the epigenetic marker set for patient stratification in routine clinical diagnostics.

Maternal Prenatal Anxiety and the Fetal Origins of Epigenetic Aging.

BACKGROUND: The fetal origins of mental health is a well-established framework that currently lacks a robust index of the biological embedding of prenatal adversity. The Pediatric-Buccal-Epigenetic (PedBE) clock is a novel epigenetic tool that associates with aspects of the prenatal environment, but additional validation in longitudinal datasets is required. Likewise, the relationship between prenatal maternal mental health and the PedBE clock has not been described. METHODS: Longitudinal cohorts from the Netherlands (Basal Influences on Baby Development [BIBO] n = 165) and Singapore (Growing Up in Singapore Towards Healthy Outcomes [GUSTO] n = 340) provided data on prenatal maternal anxiety and longitudinal assessments of buccal cell-derived genome-wide DNA methylation assessed at 6 and 10 years of age in BIBO, and at 3, 9, and 48 months of age in GUSTO. Measures of epigenetic age acceleration were calculated using the PedBE clock and benchmarked against an established multi-tissue epigenetic predictor. RESULTS: Prenatal maternal anxiety predicted child PedBE epigenetic age acceleration in both cohorts, with effects largely restricted to males and not females. These results were independent of obstetric, socioeconomic, and genetic risk factors, with a larger effect size for prenatal anxiety than depression. PedBE age acceleration predicted increased externalizing symptoms in males from mid- to late childhood in the BIBO cohort only. CONCLUSIONS: These findings point to the fetal origins of epigenetic age acceleration and reveal an increased sensitivity in males. Convergent evidence underscores the societal importance of providing timely and effective mental health support to pregnant individuals, which may have lasting consequences for both mother and child.

Brush swab as a noninvasive surrogate for tissue biopsies in epigenomic profiling of oral cancer.

BACKGROUND: Oral squamous cell carcinoma (OSCC) has poor survival rates. There is a pressing need to develop more precise risk assessment methods to tailor clinical treatment. Epigenome-wide association studies in OSCC have not produced a viable biomarker. These studies have relied on methylation array platforms, which are limited in their ability to profile the methylome. In this study, we use MethylCap-Seq (MC-Seq), a comprehensive methylation quantification technique, and brush swab samples, to develop a noninvasive, readily translatable approach to profile the methylome in OSCC patients. METHODS: Three OSCC patients underwent collection of cancer and contralateral normal tissue and brush swab biopsies, totaling 4 samples for each patient. Epigenome-wide DNA methylation quantification was performed using the SureSelectXT Methyl-Seq platform. DNA quality and methylation site resolution were compared between brush swab and tissue samples. Correlation and methylation value difference were determined for brush swabs vs. tissues for each respective patient and site (i.e., cancer or normal). Correlations were calculated between cancer and normal tissues and brush swab samples for each patient to determine the robustness of DNA methylation marks using brush swabs in clinical biomarker studies. RESULTS: There were no significant differences in DNA yield between tissue and brush swab samples. Mapping efficiency exceeded 90% across all samples, with no differences between tissue and brush swabs. The average number of CpG sites with at least 10x depth of coverage was 2,716,674 for brush swabs and 2,903,261 for tissues. Matched tissue and brush swabs had excellent correlation (r = 0.913 for cancer samples and r = 0.951 for normal samples). The methylation profile of the top 1000 CpGs was significantly different between cancer and normal samples (mean p-value = 0.00021) but not different between tissues and brush swabs (mean p-value = 0.11). CONCLUSIONS: Our results demonstrate that MC-Seq is an efficient platform for epigenome profiling in cancer biomarker studies, with broader methylome coverage than array-based platforms. Brush swab biopsy provides adequate DNA yield for MC-Seq, and taken together, our findings set the stage for development of a non-invasive methylome quantification technique for oral cancer with high translational potential.

MicroRNAs and periodontal disease: a qualitative systematic review of human studies.

PURPOSE: MicroRNAs (miRNAs) are epigenetic post-transcriptional regulators that modulate gene expression and have been identified as biomarkers for several diseases, including cancer. This study aimed to systematically review the relationship between miRNAs and periodontal disease in humans, and to evaluate the potential of miRNAs as diagnostic and prognostic biomarkers of disease. METHODS: The review was conducted following the Preferred Reporting Items for Systematic Reviews and Meta-Analysis guidelines (reference number CRD42020180683). The MEDLINE, Scopus, Cochrane Library, Embase, Web of Science, and SciELO databases were searched for clinical studies conducted in humans investigating periodontal diseases and miRNAs. Expression levels of miRNAs across the different groups were analysed using the collected data. RESULTS: A total of 1,299 references were identified in the initial literature search, and 23 articles were finally included in the review. The study designs were heterogeneous, which prevented a meta-analysis of the data. Most of the studies compared miRNA expression levels between patients with periodontitis and healthy controls. The most widely researched miRNA in periodontal diseases was miR-146a. Most studies reported higher expression levels of miR-146a in patients with periodontitis than in healthy controls. In addition, many studies also focused on identifying target genes of the differentially expressed miRNAs that were significantly related to periodontal inflammation. CONCLUSIONS: The results of the studies that we analysed are promising, but diagnostic tests are needed to confirm the use of miRNAs as biomarkers to monitor and aid in the early diagnosis of periodontitis in clinical practice.

Evaluation of the Diagnostic Potential of Candidate Hypermethylated Genes in Epithelial Ovarian Cancer in North Indian Population.

Most ovarian cancers, despite improvement in management of cancer, are still diagnosed at an advanced stage. Early detection plays an essential role in reducing ovarian cancer mortality and, therefore, is critically needed. Liquid biopsies-based approaches hold significant promise for cancer detection. The present study investigates a panel of epigenetic biomarkers for the detection of epithelial ovarian cancer. A qPCR assay has been developed based on the assessment of DNA methylation markers in circulating cell-free DNA as a minimally invasive tool. Herein, the promoter methylation of seven ovarian cancer-specific genes (RASSF1A, DAPK1, SOX1, HOXA9, HIC1, SPARC, and SFRP1) was analyzed quantitatively in 120 tissue samples by MethyLight assay. The best-performing genes were further evaluated for their methylation status in 70 matched serum cell-free DNA of cancerous and non-cancerous samples. Additionally, DNA methylation patterns of these best-performing genes were validated by clonal bisulfite sequencing. The ROC (Receiver-operator characteristic) curves were constructed to evaluate the diagnostic performances of both individual and combined gene panels. The seven candidate genes displayed a methylation frequency of 61.0-88.0% in tissue samples. The promoter methylation frequencies for all the seven candidate genes were significantly higher in cancer samples than in normal matched controls. In tissue samples, the multiplex MethyLight assay for HOXA9, HIC1, and SOX1 were the best performing gene panels in terms of sensitivity and specificity. The three best-performing genes exhibited individual frequencies of 53.0-71.0% in serum CFDNA, and the multiplex assay for these genes were identified to discriminate serum from cancer patients and healthy individuals (area under the curve: HOXA9+HIC1 = 0.95, HIC1+SOX1 = 0.93 and HOXA9+SOX1 = 0.85). The results of MethyLight showed high concordance with clonal bisulfite sequencing results. Individual genes and combined panel exhibited better discriminatory efficiencies to identify ovarian cancer at various stages of disease when analyzed in tissue and serum cell-free DNA. We report a qPCR-based non-invasive epigenetic biomarker assay with high sensitivity and specificity for OC screening. Our findings also reveal the potential utility of methylation-based detection of circulating cell-free tumor DNA in the clinical management of ovarian cancer.

A DNA Methylation-Based Gene Signature Can Predict Triple-Negative Breast Cancer Diagnosis.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer (BC) subtype and lacks targeted treatment. It is diagnosed by the absence of immunohistochemical expression of several biomarkers, but this method still displays some interlaboratory variability. DNA methylome aberrations are common in BC, thereby methylation profiling could provide the identification of accurate TNBC diagnosis biomarkers. Here, we generated a signature of differentially methylated probes with class prediction ability between 5 non-neoplastic breast and 7 TNBC tissues (error rate = 0.083). The robustness of this signature was corroborated in larger cohorts of additional 58 non-neoplastic breast, 93 TNBC, and 150 BC samples from the Gene Expression Omnibus repository, where it yielded an error rate of 0.006. Furthermore, we validated by pyrosequencing the hypomethylation of three out of 34 selected probes (FLJ43663, PBX Homeobox 1 (PBX1), and RAS P21 protein activator 3 (RASA3) in 51 TNBC, even at early stages of the disease. Finally, we found significantly lower methylation levels of FLJ43663 in cell free-DNA from the plasma of six TNBC patients than in 15 healthy donors. In conclusion, we report a novel DNA methylation signature with potential predictive value for TNBC diagnosis.