DNA methylation markers in esophageal cancer: an emerging tool for cancer surveillance and treatment

Abstract

Esophageal carcinoma (EC) is one of the most pervasive cancers in the world, with upwards of 500,000 new diagnoses, annually. Despite its prominence, advancements in the detection and treatment of EC have been marginal over the past 30 years and the survival rate continues to stay below 20%. This is due to the uncommonly heterogeneous presentation of EC which presents unprecedented challenges in improving patient survival and quality of care. However, distinct epigenetic alterations to the DNA methylome may provide an avenue to drastically improve the detection and treatment of EC. Specifically, the creation of novel biomarker panels that consist of EC-specific methylation markers have shown promise as a potential alternative to the more invasive, contemporary diagnostic methods. Additionally, growing insight into the biological and clinical properties of EC-specific methylation patterns have opened a window of opportunity for enhanced treatment; of growing interest is the application of “DNMT inhibitors” - a class of drugs which inhibit excessive methylation and have been shown to re-sensitize chemoresistant tumors. Here we provide a comprehensive review of the current advancements in EC DNA methylation to underscore a potential approach to its detection and treatment.

Introduction

Every year, over 500,000 people worldwide are diagnosed with esophageal carcinoma (EC), with approximately the same number of EC-related deaths. The two major subtypes of EC are esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC). While ESCC, commonly seen in Africa, Asia and South America, is the predominant form of EC, the number of EAC cases has increased 600-800% in developed, western countries over the past 30 years. Due to this increase, EC has become the 6^th most common cause of cancer-related deaths in the United States [1,2]. Both ESCC and EAC are markedly prevalent in men over women, with men accounting for 70% of all EC cases worldwide [3]. In contrast, obesity and gastroesophageal reflux disease (GERD) has been strongly linked to an increased risk for EAC, while smoking and alcohol consumption pose an increased risk for ESCC [4].

Currently, given the high mortality rate of EC and the inefficacy of existing treatments, prevention and early detection continue to yield the best chances of survival. The defining precursors for EAC and ESCC are Barrett’s Esophagus (BE) and squamous dysplasia, respectively, with BE representing a 50 to 100-fold increased risk for carcinogenesis [5]. Moreover, these pre-malignant lesions provide an opportunity for the early detection and treatment of ECs, given their locations. Both BE and squamous dysplasia occupy the same regions as their associated malignancies: BE is found to be at the gastroesophageal junction (GEJ) and in the distal third of the esophagus, while squamous dysplasia is found in the proximal two-thirds of the esophagus [3,6]. At present, the gold standard for EC screening is endoscopy, with biopsy for iodine staining [1]; however, while screening for these pre-malignant lesions offer some forewarning of subsequent EC, BE alone is not an absolute indicator of malignancy. Moreover, such invasive and financially burdensome procedures call into question the feasibility of widespread endoscopic screening for EC. Specifically, given the large proportion of ESCC patients residing in impoverished parts of the world, labor-intensive and costly screening methods will only marginally help reduce the mortality rate of EC. For this reason, EC typically remains undiagnosed until advanced stage disease, when the overall survival is typically ≤1 year [1]. Despite significant advancements in medicine and cancer biology over the past several decades, treatment outcomes for EC have only seen marginal improvements, keeping the 5-year survival rate at 15-20% [1].

Despite our understanding of pre-existing conditions which pose increased risks for EC carcinogenesis, the absence of robust molecular markers have largely contributed to the marginal advancements in EC treatments. EC’s continuously low survival rate can also be attributed to its exceptionally heterogenous presentation. Thousands of genomic aberrations have been linked to EC [7,8]. In fact, the extent of EC’s heterogeneity is not only seen among different patients, but also, differing genomic alterations have been reported to be present in different sections of the same tumor [9-11]. This degree of heterogeneity has remained a foremost impediment for researchers who try to stratify risk levels associated with disease progression and identify reliable prognostic indicators. Therefore, the establishment of non-interventional, economically feasible screening methods in conjunction with dependable biomarkers are of paramount importance in mitigating EC’s lethality.

DNA methylation

Epigenetic markers on DNA are heritable modifications that alter gene function or activity, yet do not change the underlying DNA sequence. One prominent marker in EC is DNA methylation - the process by which Cytosine residues are methylated at the C5 position, therein becoming 5-methylcytosine. DNA methylation is essential for myriad functions, including mammalian development, and is catalyzed by several different proteins, termed epigenetic writers [12], in the DNA methyltransferase (DNMT) family of enzymes. In general, DNMT3a/b are responsible for de novo methylation, while DNMT1 maintains existing DNA methylation by copying methylation patterns onto newly synthesized strands during DNA replication [13]. While DNA methylation is paramount in modulating gene expression across a wide variety of cells, its dysregulation has been linked to carcinogenesis, and is classified into two categories: hypermethylation and hypomethylation. Hypermethylation is associated with gene repression, while hypomethylation is correlated to increased gene expression [14]. DNA methylation changes alter the recruitment of epigenetic regulators and transcription factors to their binding sites [15]. In EC, the two predominant forms of dysregulated methylation patterns are global hypomethylation, and CpG-specific hypermethylation [16,17] (Figure 1).

Figure 1.

Aberrant DNA methylation in carcinogenesis. DNA methylation, catalyzed by DNMT family enzymes, alter gene function or activity in esophageal cancer. DNA methylation, mainly classified into hypermethylation and hypomethylation, in esophageal cancer alter different gene function or activity. After the interaction of DNMT and DNA binding sites, global hypomethylation causes the enhanced activity of the genes, such as LINE-1, and CpG-specific hypermethylation is correlated to gene expression reduction, such as MGMT and CDKN2A.

Over the past several decades, multiple studies have shown that global hypomethylation plays a significant role in carcinogenesis, through several Mechanisms [18-21] (Figure 1). Namely, the hypomethylation of parasitic, repetitive DNA sequences, such as retrotransposons, or of proto-oncogenes has pronounced deleterious effects on chromosomal stability and cellular function [22]. Further, excessive hypomethylation of centromeric and pericentromeric satellite sequences is commonly seen in a variety of tumors, and its consequential effects on chromosomal stability has been suggested to lead to aneuploidy [23]. Following its established significance in carcinogenesis, meta-analyses of global DNA hypomethylation in various cancers have suggested a link between the extent of hypomethylation and cancer stage [22,24].

In contrast to its activating counterpart, extensive DNA hypermethylation of promoter-region CpG islands has been reported to largely facilitate tumorigenesis by repressing tumor suppressor genes [25]. Numerous studies have since shown the degree to which hypermethylation promotes tumorigenesis. One example of this was shown in the promoter region of the cyclin-dependent kinase (CDK) inhibitor gene, p16^INK4A, whereby hypermethylation is observed to occur in the pre-cancerous stages of tumorigenesis [26-29]. Another mechanism by which DNA hypermethylation results in aberrant gene expression is through C>T point mutations, via spontaneous deamination of 5-methylcytosine which ultimately is replaced by thymine residues, if not repaired [30]. Such reactivating hypermethylation mutagenesis patterns are often seen in the promoter region of Telomerase Reverse Transcriptase (TERT) complex, which is found in approximately 90% of human cancers, including gastric, pancreatic, and cervical cancers [31-38]. Therefore, given the extent to which aberrant methylation patterns - both of hypo and hypermethylation - influence the onset of carcinogenesis, developing better methods for monitoring alterations to the methylome will play a pivotal role in preventing cancer progression.

Screening for esophageal carcinoma

Current techniques used for the screening of EC are similar and not sufficient for the diagnosis of precancerous lesions. One example being traditional white-light endoscopy (WLE), which is routinely used for the detection of invasive esophageal carcinomas, and even low-grade BE dysplasia. However, WLE is not capable of detecting esophageal squamous dysplasia [39,40]. A common and inexpensive alternative to WLE is chromoendoscopy, which is sensitive enough to detect precursor lesions, but has insufficient and inconsistent specificity (37%-82%) for squamous dysplasia [41-44]. Other conventional endoscopic methods include transnasal endoscopy, microendoscopy, and endocytoscopy - all of which consist of flexible probes surveying the esophagus (Table 1).

Table 1.

Comparison of the commonly used screen methods for early esophageal carcinomas

METHOD	PROCEDURE	BENEFITS	DRAWBACKS	REF.
WHITE-LIGHT ENDOSCOPY	Endoscope with high-resolution imaging - may be done with endoluminal biopsy	Is sensitive enough to detect BE-LGD	Can’t detect ESCC precursor lesions; expensive; invasive	[116]
LUGOL CHROMOENDOSCOPY	Application of colored dyes to esophagus for targeted biopsy	Inexpensive; high sensitivity	Inconsistent specificity for squamous dysplasia	[117]
TRANSNASAL ENDOSCOPY	Nasal esophageal intubation with optic scope	Economical; less invasive; no need for general anesthesia	Conscious sedation poses increased risk for complication	[118]
MICROENDOSCOPY	Confocal laser endomicroscopy of targeted biopsy tissue	Higher sensitivity and specificity, relative to conventional microscopy methods	Not economically feasible - can cost thousands of dollars	[119]
ENDOCYSTOCOPY	Ultra-magnification of stained, or in-vivo, epithelial tissue	High sensitivity/specificity	Not readily available; expensive; lack of standardized criteria for diagnosis leads to ambiguous results	[120]
CYTOSPONGE	Ingested sponge capsule is retrieved from patient after 5 minutes wherein the apparatus scrapes cells from the epithelium	Very high sensitivity/specificity; non-invasive; inexpensive; convenient; can collect 500,000 cells for epigenetic analysis therein increasing reproducibility and robustness	Still requires traditional endoscopic methods, following a positive result; sensitivity is still not 100%	[90]
ESOPHACAP	Ingested sponge is retrieved after several minutes whereupon removal, cells are extracted	Highest sensitivity/specificity; 1,000,000 cells are collected; inexpensive; convenient; non-invasive	Still requires traditional endoscopic methods, following a positive result	[91]

To detect aberrant methylation in patients with EC, typically one of the aforementioned endoscopic methods would be used for tissue biopsy, though newer, non-invasive methods such as the Cytosponge or Esophacap are being used to collect cells from the esophageal mucosa. Subsequently, a variety of assays, generally beginning with bisulfite conversion, could be performed to detect methylation [45]. Bisulfite conversion works by deaminating unmethylated cytosine residues to uracil while leaving methylate cytosine residues alone, therein highlighting the presence and location of DNA methylation [46,47]. Following bisulfite conversion, a number of different assays can be performed, including sanger or pyrosequencing [48]. Other popular methods of DNA methylation detection include array-based platforms and Methyl-cytosine based immunoprecipitation (IP) followed by sequencing [49,50] (Figure 2).

Figure 2.

Methylation-based screening for esophageal carcinoma. Tissue biopsy can be obtained by endoscopic methods. After sample collection, it will be directly used for DNA extraction and bisulfite conversion, which could highlight the presence and location of DNA methylation. Several assays can be performed for methylation detection, such as sanger-sequencing or pyrosequencing.

Potential applications of DNA methylation markers in EC

The importance of DNA methylation in cancer was first highlighted in 1983 when significant hypomethylation was observed in cancer cells, in contrast to the surrounding healthy cells [51]. Since then, deleterious epigenetic modifications in ECs have been investigated for a variety of reasons. At present, limitations in our understanding of EC biology has been a significant roadblock in developing effective treatment regimens, although existing therapeutics have yielded some degree of success. As a result, epigenetic signatures in EC have been the focus of biomarker research, offering the opportunity to serve not only in a diagnostic capacity, but also for the monitoring and prognostication of disease progression (progression markers), survival prognosis (prognostic markers), and likelihood of response to therapy (predictive markers). From scrutinizing methylomic changes in EC, several genes that present aberrant expression levels have been identified as preliminary inducers of carcinogenesis from BE to EAC, and can be seen before malignant histologic changes are observed [52]. Moreover, as the degree of DNA methylation becomes more prominent throughout the course of disease progression, clinicians are then able to more effectively implement an appropriate treatment plan that is specifically tailored to each patient.

While the primary focus of methylation markers in EC has been for surveillance purposes, the stratification of EC’s based upon distinct molecular signatures has shown promise in working towards delivering targeted therapeutics. Specifically, this classification of ECs into categorical subtypes has allowed clinicians to treat ESCC and EAC by addressing the root cause of abnormal gene activity [53]. For example, hypermethylated carcinomas appear to be sensitive to treatment with DNA methyltransferase and topoisomerase I inhibitors, while global hypomethylation may be sensitive to treatment with CDK2 inhibitors [54]. Although promising, further rigorous clinical validation is necessary to confirm the reliability of universal epigenetic alterations that are indicative of EC and its subsequent progression.

Ongoing methylation marker discovery and validation for esophageal carcinoma and precursor lesions

From data compiled by The Cancer Genome Atlas (TCGA) team, over 8,300 genes were found to be mutated across 165 cases of EAC [7]. Moreover, Bandla et al. reported the significance of mutational load in differentiating EAC from its precursor, BE [55]. Specifically, the buildup of epigenetic and genetic modifications in several genes - including TP53, CDKN2A, CTNNB1, and APC - that are commonly seen in EAC have also been reported to be present in BE, albeit less frequently [56-61]. The progressive accumulation of these mutations has therefore allowed for the stratification of disease into stages; this multistep progression starts with BE, then transitioning to low-grade dysplasia (LGD), subsequent high-grade dysplasia (HGD), and ultimately EAC [62,63] (Figure 3). However, given the level of heterogeneity and complexity in EC, a meticulous validation process is essential for confirming the robustness of molecular signatures which are indicative of the presence of ECs.

Figure 3.

Application of DNMT inhibitor therapeutics in halting progression of EAC. During the progression of esophageal carcinoma, DNA methylation, catalyzed by DNMT, was modified on several genes, causing aberrant expression and activity. DNMT inhibitor treatment could reduce the degree/extent of DNA methylation and increase the expression of tumor suppressor genes, and thus improve clinical outcomes.

In an effort to address this, Alvi et al. used an array-based approach and formed a four-marker panel (PIGR, RIN2, GJA12, and SLC22A18) that was able to distinguish between the presence of HGD/EAC and BE, and enhanced classification of disease-related risk, based upon the degree of methylation in this panel [64]. In a retrospective study, using a cohort consisting of 60 BE patients, 36 patients with dysplastic BE, and 90 with HGD/early EAC, this panel was validated by pyrosequencing and was shown to have a specificity of 97% and a sensitivity of 94%. In a broader approach, the Esophageal Cancer Clinical and Molecular Stratification (OCCAMS) consortium analyzed the results from whole-genome sequencing of 129 EAC cases and were able to categorize the mutational profiles into 3 distinct EAC groups based upon differing genomic mutations. These findings were substantiated in a subsequent study which analyzed the mutational profiles of another cohort, consisting of 87 patients [65]. This study served as the basis for Jammula et al.’s epigenetic analyses on over 300 BE and EAC cases; the results of which were consolidated with corresponding genomic and transcriptomic profiles to present a comprehensive analysis to confidently discern BE from EAC [54]. The data gathered allowed for the classification of EAC and BE tissues to be stratified into 4 subtypes; among the 4 subtypes, 3 involve aberrant methylation patterns that have been suggested to be clinically relevant for treatment regimen planning. Subtype 1 tissue displayed hypermethylation in the CpG islands of noncoding regions for genes necessary for DNA repair. Subtype 2 tissue likewise exhibited hypermethylation; however, also displayed hypomethylation in genes required for cell metabolism and ATP synthesis. The last subtype involving anomalous methylation signatures, subtype 4, presented extensive hypomethylation which resulted in significant structural variation.

Although having different etiologies, many of the aberrant methylation signatures found in EAC are also commonly seen in ESCC, further compounding the level of difficulty in biomarker validation. Moreover, ESCC presents the same multistep progression as is seen in EAC (non-dysplastic, LGD, HGD, carcinoma), wherein mutational load and accumulation of epigenetic alterations can assist with risk stratification for survivability and probability of carcinogenesis [66-71]. An example of this is the promoter hypermethylation of several tumor suppressor genes and DNA repair genes (MGMT, p14, p16) that have been found to be present in EAC as well as in dysplastic, ESCC precursor lesions [72-74]. Specifically, CDKN2A (official symbol for p16) hypermethylation is found to be present in up to 88% of ESCC tumors, and is indicative of an invasive phenotype [75-83]. Other well-known oncogenes such as KRAS, IGF1R, CDK6, and EGFR are also overexpressed in both EAC and ESCC, at similar frequencies. Additionally, the proximity in which these two malignancies can present, within the esophageal tract, may hinder the ability to accurately discern between histopathological differences.

To better discriminate between carcinomas in adjacent tissue, Pu et al. discovered and validated a 5-marker panel that showed significant CpG island hypermethylation in ESCC tissue, relative to EAC tissue. Three of these markers are in the promoter region of genes (STK3, ZNF418, and ZNF542) [84,85]. Additionally, Agrawal et al. highlighted distinct spectra in C:G>T:A mutation profiles in EACs (46%) vs. ESCCs (35%), with A:T>C:G substitutions more commonly found in EAC as opposed to a higher incidence of C:G>G:C substitutions and indels found in ESCC [86]. A more recent molecular distinction was shown in the overexpression of several genes, namely SOX2, CCND1, and/or TP63 are more frequently seen in ESCC, while GATA6, GATA4, and ERBB2 are typically representative of EAC [87]. In a different study, Lu et al. analyzed RNA-seq and methylation profiles in ESCC and found five candidate genes (ZNF608, SLC5A10, ZNF69, SPIN3, and ABCD1) with aberrant methylomic signatures that may potentially serve as prognostic makers for ESCC (See Table 2) [88]. Furthermore, as more molecular markers for EC are being discovered and validated, aberrant DNA methylation is a promising method by which both EAC and ESCC can be not only accurately diagnosed, but further categorized based upon a host of factors affecting clinical outcomes.

Table 2.

Promising Epigenomic Biomarkers for EC

Marker	Function	Methylation Status	Malignancy	Reference
HER2 (ERBB2)	Cell differentiation; proliferation; suppresses apoptosis	Hypo	EAC	[110]
PD-L1	Immune checkpoint, signals for apoptosis	Hypo	ESCC	[111]
CDKN2A	Tumor suppressor gene	Hyper	EAC/ESCC	[73]
ABCD1	ABC transporter protein for fatty acids	Hypo	ESCC	[88]
SPIN3	Tumor suppressor gene	Hyper	ESCC	[88]
TP63	Antagonist of pro-apoptotic genes	Hypo	ESCC	[87]
GATA4/6	Transcription factor	Hypo	EAC	[87]
SOX2	Transcription factor; cell renewal	Hypo	ESCC	[87]
LINE-1	Transposable element	Hypo	ESCC	[99]
KRAS	Part of RAS/MAPK pathway; cell proliferation	Hypo	EAC/ESCC	[84]

Benefits and limitations of methylation markers in the detection and surveillance of EC

Although more progress still needs to be made, integrating methylation markers into routine EC screening has numerous potential benefits, including the potential for increased sensitivity and specificity over current methods [89]. Additionally, methylation marker panels that are currently being studied allow for the utilization of non-invasive methods of sample collection. In 2018, Chettouh et al. used a four-marker panel comprised of TFPI2, TWIST1, ZNF569, and ZNF345 to screen for BE, and were able to use an inexpensive, non-endoscopic Cytosponge collection apparatus [90]. Moreover, Wang et al. used a non-invasive Esophacap to collect esophageal mucosal cells and were able to discern healthy controls from BE samples using a five-marker panel (AKAP12, NELL1, HPP1, p16, and TAC1) with a specificity of 92.8% and a sensitivity of 78.6% [91]. Given the potential of reduced costs, reduced need for invasive endoscopic procedures, and increased sensitivity and specificity, the implementation of robust methylation-marker panels for the detection and surveillance of ECs shows promise as an alternative to conventional methods.

On the other hand, in spite of the progress made in identifying and validating methylation markers in EC, current methodologies also have some limitations. Of note, there is interobserver variability in diagnosing BE-LGD and its subsequent progression to carcinoma, which makes it difficult to standardize the results of biomarker studies and to identify a potential universal methylation marker panel. To help address this issue, investigation of the epigenomic alterations in esophageal cells during BE and EC carcinogenesis has shown increasing promise as a method through which objective analysis can be conducted. In doing so, many have used in-vitro cell, and 3D, culture models and in vivo animal models; however, these studies have received criticism for large discrepancies between these models and human EC pathobiology [92-98]. In addition to interobserver variations among clinicians and scientists, the exceptional degree of heterogeneity seen in ECs further complicates the task of finding reliable indicators of malignancy, or precursor lesions. For example, hypomethylation of the repetitive long interspersed transposable element (LINE), LINE-1, has been proposed as a biomarker for ESCC that is strongly correlated with high risk for progression and poor prognosis. However, studies on LINE-1 in EC vary greatly in their analyses of the proportion of hypomethylation required (25-92% increase) to exercise deleterious affect [99-102]. Therefore, it is paramount that improvements in methylation biomarkers be made before being put into practice.

Current directions in biomarker-related treatments for EC

Although many therapeutic interventions for EC are either still in development or have yielded modest success, new insight into EC methylomes have provided potential therapeutic targets. Of growing interest, the employment of DNMT inhibitors has shown promise in combination therapies for the treatment of chemoresistant tumors. Specifically, the methylation-induced repression of specific genes, that would otherwise sensitize tumor cells to chemotherapy, nulls the cytotoxic effects of conventional therapies, thereby rendering these treatments ineffective. Additionally, as previously discussed, many tumor suppressor genes (TSGs) are hypermethylated during EC carcinogenesis. This epigenetic repression has consequently led to recent investigation into the application of DNMT inhibitors (Figure 3).

Currently, the most well-known DNMT inhibitor, albeit still not fully understood, is 5-azacitidine which is an FDA approved nucleoside analog that is capable of inducing hypomethylation and has been shown to improve EC patient survivability [103,104]. In a cohort of 12 esophageal/gastric adenocarcinoma patients, Schneider et al. found that treatment with 5-azacitidine (V, 75 mg/m²) for 3-5 days prior to chemotherapy resulted in reactivation of hypermethylated TSGs, which may result in hypomethylation-induced chemoresensitization, ultimately improving clinical outcomes of subsequent chemotherapy and resection of residual tumors [105]. In a similar study, Fu et al. found 5-azacitidine (V, 75 mg/m²) for five days at least partially restored chemotherapeutic efficacy in chemoresistant ovarian cancers [106]. In addition to 5-azacitidine, the other FDA approved DNMT inhibitor is decitabine, the deoxy derivative of 5-azacitidine, which yields comparable results and works in similar fashion: incorporating itself onto DNMT DNA strands during replication wherein it acts as a chain terminator to inhibit activity [107-109]. Although there are currently not many therapeutics that directly act on the epigenomic changes caused by EC carcinogenesis, advancements in EC biology and epigenomics have allowed clinicians to better formulate personalized treatment regimens using current therapeutics.

Aside from the direct therapeutic targeting of epigenetic writers, significant advancements in biomarker-based immunotherapy have been instrumental in directing future research efforts. In particular, excessive hypomethylation of well-known biomarkers, such as PD-L1 and HER2 have contributed to their overexpression within the tumor microenvironment, and thus, its immunosuppressive properties [110,111]. Although further research needs to be done to improve efficacy, clinical trials on Trastuzumab, a humanized HER-2 monoclonal antibody, have improved progression-free survival in EAC patients, when combined with chemotherapy [112,113]. Moreover, myriad studies and clinical trials on the efficacy of a PD-L1 blockade therapy, such as Nivolumab and Keytruda, have yielded objective response rates of up to 20%, adding as much as 6 months to overall survival in ESCC patients [114,115]. It was also noted that overexpression of PD-L1 is correlated with microsatellite instability (MSI) and a DNA mismatch repair (MMR) deficiency. As a result, in conjunction with the PD-L1 and HER2 biomarkers, the National Comprehensive Cancer Network (NCCN) has also listed surveillance of MSI/MMR status to its pathologic biomarker testing guidelines. As previously mentioned, extensive DNA hypomethylation of centromeric and pericentromeric satellite sequences have significant deleterious effects on chromosomal stability and sequence integrity. Therefore, the combination of these suggested biomarkers, per NCCN guidelines, can serve in both diagnostic and therapeutic capacities, and are promising advancements in biomarker research for EC.

Conclusion

EC is one of the deadliest forms of cancer, with 5-year survival rate of less than 20%. This is largely due to asymptomatic patients who remain undiagnosed until late-stage carcinoma, when the survival rate is exceedingly low. This review addresses the significance of aberrant DNA methylation in ECs and the potential impact of advancing our understanding of methylation markers throughout the various stages of EC carcinogenesis, from non-dysplastic precursor lesions to invasive carcinoma. Moreover, studies over the past decade have revealed the applications of methylation markers in EC to be beneficial in diagnostic, prognostic, and therapeutic capacities. Of growing interest is the combination of distinct epigenomic markers to form “panels”, therein enhancing the accuracy of diagnostic and prognostic screening. Further, such panels have been helpful, thus far, in differentiating between different malignancies that would otherwise be difficult to discern due to proximity or histological presentation (See Table 2). Therefore, advancements in this field may not only improve overall survival, due to early detection, but may ultimately reveal novel therapeutic targets for improved treatment. However, the fact remains that despite improvements in methylome-based approaches for the detection, surveillance, and therapeutics for EC, challenges such as overcoming extensive heterogeneity and interobserver variability remain and underscore the importance of continuing to better understand DNA methylation in EC and to work towards improving this platform.

Disclosure of conflict of interest

None.