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.

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.

The Contribution of Epigenetics to Cancer Immunotherapy.

Effective anticancer immunotherapy treatments constitute a qualitative leap in cancer management. Nonetheless, not all patients benefit from such therapies because they fail to achieve complete responses, suffer frequent relapses, or develop potentially life-threatening toxicities. Epigenomic signatures in immune and cancer cells appear to be accurate and promising predictors of patient outcomes with immunotherapy. In addition, combined treatments with epigenetic drugs can exploit the dynamic nature of epigenetic changes to potentially modulate responses to immunotherapy. Candidate epigenetic biomarkers may provide a rationale for patient stratification and precision medicine, thus maximizing the chances of treatment success while minimizing unwanted effects. We present a comprehensive up-to-date view of potential epigenetic biomarkers in immunotherapy and discuss their advantages over other indicators.

Stratifying Cumulus Cell Samples Based on Molecular Profiling to Help Resolve Biomarker Discrepancies and to Predict Oocyte Developmental Competence.

To increase the efficiency of assisted reproductive techniques (ART), molecular studies have been performed to identify the best predictive biomarkers for selecting the most suitable germ cells for fertilization and the best embryo for intra-uterine transfer. However, across different studies, no universal markers have been found. In this study, we addressed this issue by generating gene expression and CpG methylation profiles of outer cumulus cells obtained during intra-cytoplasmic sperm injection (ICSI). We also studied the association of the generated genomic data with the clinical parameters (spindle presence, zona pellucida birefringence, pronuclear pattern, estrogen level, endometrium size and lead follicle size) and the pregnancy result. Our data highlighted the presence of several parameters that affect analysis, such as inter-individual differences, inter-treatment differences, and, above all, specific treatment protocol differences. When comparing the pregnancy outcome following the long protocol (GnRH agonist) of ovarian stimulation, we identified the single gene markers (NME6 and ASAP1, FDR < 5%) which were also correlated with endometrium size, upstream regulators (e.g., EIF2AK3, FSH, ATF4, MKNK1, and TP53) and several bio-functions related to cell death (apoptosis) and cellular growth and proliferation. In conclusion, our study highlighted the need to stratify samples that are very heterogeneous and to use pathway analysis as a more reliable and universal method for identifying markers that can predict oocyte development potential.

The "-OMICS" facet of melanoma: Heterogeneity of genomic, proteomic and metabolomic biomarkers.

In the recent decade, cutting edge molecular and proteomic analysis platforms revolutionized biomarkers discovery in cancers. Melanoma is the prototype with over 51,100 biomarkers discovered and investigated thus far. These biomarkers include tissue based tumor cell and tumor microenvironment biomarkers and circulating biomarkers including tumor DNA (cf-DNA), mir-RNA, proteins and metabolites. These biomarkers provide invaluable information for diagnosis, prognosis and play an important role in prediction of treatment response. In this review, we summarize the most recent discoveries in each of these biomarker categories. We will discuss the challenges in their implementation and standardization and conclude with some perspectives in melanoma biomarker research.

5-Hydroxymethylcytosine as a clinical biomarker: Fluorescence-based assay for high-throughput epigenetic quantification in human tissues.

Epigenetic transformations may provide early indicators for cancer and other disease. Specifically, the amount of genomic 5-hydroxymethylcytosine (5-hmC) was shown to be globally reduced in a wide range of cancers. The integration of this global biomarker into diagnostic workflows is hampered by the limitations of current 5-hmC quantification methods. Here we present and validate a fluorescence-based platform for high-throughput and cost-effective quantification of global genomic 5-hmC levels. We utilized the assay to characterize cancerous tissues based on their 5-hmC content, and observed a pronounced reduction in 5-hmC level in various cancer types. We present data for glioblastoma, colorectal cancer, multiple myeloma, chronic lymphocytic leukemia and pancreatic cancer, compared to corresponding controls. Potentially, the technique could also be used to follow response to treatment for personalized treatment selection. We present initial proof-of-concept data for treatment of familial adenomatous polyposis.

Epigenetic biomarkers indicate islet cell death in xenotransplantation.

BACKGROUND: Xenotransplantation of porcine islets has emerged in recent decades as a potential treatment for type 1 diabetes (T1D). Current methods of detection, indicative of successful engraftment, occur downstream of actual islet death. Epigenetic biomarkers can be detected in circulating cell-free DNA (cfDNA) to provide an earlier indication of graft dysfunction. AIMS: The present study identified a biomarker of islet death using differential methylation of the insulin gene, INS, originating from ß-cells in porcine islets. MATERIALS & METHODS: Pyrosequencing primers specific for porcine INS were designed to quantify hypomethylation along 12 cysteine-guanine dinucleotide (CpG) sites, including three sites in the cyclic adenosine monophosphate (cAMP) response element (CRE) binding protein 2 (CRE2) binding region of the 5' untranslated region (UTR) and nine sites within intron 2. RESULTS: PCR amplification of bisulfite-converted DNA combined with pyrosequencing data support the conclusion that hypomethylated porcine INS is specific to islet origin. CONCLUSION: Moreover, the results of this study indicate a highly specific epigenetic biomarker, capable of detecting a single islet, supporting the measurement of cfDNA as a biomarker for transplanted islet death. Defining the epigenetic characteristics of porcine-derived islets within cfDNA will be crucial to develop a better understanding of graft survival immunology for transplantation.

Aberrant DNA Methylation in Acute Myeloid Leukemia and Its Clinical Implications.

Acute myeloid leukemia (AML) is a heterogeneous disease that is characterized by distinct cytogenetic or genetic abnormalities. Recent discoveries in cancer epigenetics demonstrated a critical role of epigenetic dysregulation in AML pathogenesis. Unlike genetic alterations, the reversible nature of epigenetic modifications is therapeutically attractive in cancer therapy. DNA methylation is an epigenetic modification that regulates gene expression and plays a pivotal role in mammalian development including hematopoiesis. DNA methyltransferases (DNMTs) and Ten-eleven-translocation (TET) dioxygenases are responsible for the dynamics of DNA methylation. Genetic alterations of DNMTs or TETs disrupt normal hematopoiesis and subsequently result in hematological malignancies. Emerging evidence reveals that the dysregulation of DNA methylation is a key event for AML initiation and progression. Importantly, aberrant DNA methylation is regarded as a hallmark of AML, which is heralded as a powerful epigenetic marker in early diagnosis, prognostic prediction, and therapeutic decision-making. In this review, we summarize the current knowledge of DNA methylation in normal hematopoiesis and AML pathogenesis. We also discuss the clinical implications of DNA methylation and the current therapeutic strategies of targeting DNA methylation in AML therapy.

Nutritional Factors, DNA Methylation, and Risk of Type 2 Diabetes and Obesity: Perspectives and Challenges.

A healthy diet improves life expectancy and helps to prevent common chronic diseases such as type 2 diabetes (T2D) and obesity. The mechanisms driving these effects are not fully understood, but are likely to involve epigenetics. Epigenetic mechanisms control gene expression, maintaining the DNA sequence, and therefore the full genomic information inherited from our parents, unchanged. An interesting feature of epigenetic changes lies in their dynamic nature and reversibility. Accordingly, they are susceptible to correction through targeted interventions. Here we will review the evidence supporting a role for nutritional factors in mediating metabolic disease risk through DNA methylation changes. Special emphasis will be placed on the potential of using DNA methylation traits as biomarkers to predict risk of obesity and T2D as well as on their response to dietary and pharmacological (epi-drug) interventions.

Genome Instability and γH2AX.

γH2AX has emerged in the last 20 years as a central player in the DDR (DNA damage response), with specificity for DSBs (double-strand breaks). Upon the generation of DSBs, γ-phosphorylation extends along megabase-long domains in chromatin, both sides of the damage. The significance of this mechanism is of great importance; it depicts a biological amplification mechanism where one DSB induces the γ-phosphorylation of thousands of H2AX molecules along megabaselong domains of chromatin, that are adjusted to the sites of DSBs. A sequential recruitment of signal transduction factors that interact to each other and become activated to further amplify the signal that will travel to the cytoplasm take place on the γ-phosphorylated chromatin. γ-phosphorylation is an early event in the DSB damage response, induced in all phases of the cell cycle, and participates in both DSB repair pathways, the HR (homologous recombination) and NHEJ (non-homologous end joining). Today, numerous studies support the notion that γH2AX functions as a guardian of the genome by preventing misrepaired DSB that increase the mutation load of the cells and may further lead to genome instability and carcinogenesis.

Accounting for Life-Course Exposures in Epigenetic Biomarker Association Studies: Early Life Socioeconomic Position, Candidate Gene DNA Methylation, and Adult Cardiometabolic Risk.

Recent studies suggest that epigenetic programming may mediate the relationship between early life environment, including parental socioeconomic position, and adult cardiometabolic health. However, interpreting associations between early environment and adult DNA methylation may be difficult because of time-dependent confounding by life-course exposures. Among 613 adult women (mean age = 32 years) of the Jerusalem Perinatal Study Family Follow-up (2007-2009), we investigated associations between early life socioeconomic position (paternal occupation and parental education) and mean adult DNA methylation at 5 frequently studied cardiometabolic and stress-response genes (ABCA1, INS-IGF2, LEP, HSD11B2, and NR3C1). We used multivariable linear regression and marginal structural models to estimate associations under 2 causal structures for life-course exposures and timing of methylation measurement. We also examined whether methylation was associated with adult cardiometabolic phenotype. Higher maternal education was consistently associated with higher HSD11B2 methylation (e.g., 0.5%-point higher in 9-12 years vs. ≤8 years, 95% confidence interval: 0.1, 0.8). Higher HSD11B2 methylation was also associated with lower adult weight and total and low-density lipoprotein cholesterol. We found that associations with early life socioeconomic position measures were insensitive to different causal assumption; however, exploratory analysis did not find evidence for a mediating role of methylation in socioeconomic position-cardiometabolic risk associations.

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.

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.

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 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.

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.

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 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.

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.

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.