INTRODUCTION
Esophageal cancer (EC) remains one of the most aggressive gastrointestinal cancers worldwide, with high mortality rates, primarily due to late-stage diagnosis[1,2]. Despite advancements in treatment modalities, the prognosis for patients with EC is closely tied to the stage at which the disease is detected. Unfortunately, early-stage EC often presents with nonspecific symptoms, leading to delayed diagnoses[3]. Current diagnostic methods, including double-contrast barium X-ray and endoscopy, are invasive and are often employed only when symptoms become severe[4]. Furthermore, routine endoscopic screening is not widely accepted, particularly in regions such as China, where the incidence of EC is highest[5]. This has resulted in a low rate of early detection, contributing to poor overall survival outcomes[6]. Therefore, there is an urgent need for novel diagnostic approaches that can facilitate early, noninvasive screening and improve patient prognosis.
One promising innovation in cancer diagnostics is the use of circulating tumor DNA (ctDNA), specifically focusing on DNA methylation patterns as biomarkers. The ctDNA is released into the bloodstream by tumor cells and undergoes cancer-specific genetic and epigenetic alterations, including DNA methylation, which occurs early in tumorigenesis[7]. The presence of methylation in specific gene promoters can serve as a reliable marker for cancer detection[8]. In the study by Liu et al[9], the methylation of SHOX2, SEPTIN9, EPO, and RNF180 was investigated as a tool for diagnosing esophageal adenocarcinoma and squamous cell carcinoma. The authors demonstrated that combining these four methylation markers significantly improved the diagnostic sensitivity (76.19%) and specificity (86.27%) for EC. These results highlight the potential of ctDNA methylation as an early diagnostic tool, offering a noninvasive alternative to current methods, with the added benefit of enabling real-time monitoring of tumor progression and treatment response[10].
Liquid biopsy, which uses ctDNA to detect cancer-related changes through a simple blood sample, represents a significant advance in cancer diagnostics[11,12]. Unlike traditional biopsy methods, liquid biopsy is minimally invasive and can be repeated throughout treatment to track disease progression[13,14]. The study by Liu et al[9] highlights the growing potential of liquid biopsy in clinical practice, revealing how methylation markers can enhance the diagnostic capabilities for EC. With continuous advancements in this field, liquid biopsy has the potential to revolutionize cancer screening, enabling earlier detection and more personalized treatment approaches[15]. Moving forward, integrating ctDNA methylation with other molecular and imaging biomarkers could establish a new standard for EC screening, monitoring, and treatment in clinical settings[16].
CURRENT LANDSCAPE OF DNA METHYLATION IN CANCER
Currently, research on ctDNA is advancing rapidly, establishing it as a vital tool in cancer diagnosis and monitoring. The ctDNA consists of small fragments of DNA released into the bloodstream by tumor cells[7]. The mechanisms by which ctDNA enters the circulation involve several processes, including apoptosis, necrosis, and active secretion from tumor cells. When tumor cells undergo apoptosis, they fragment their genomic DNA, resulting in the release of these small DNA fragments into the bloodstream. This ctDNA often carries tumor-specific genetic alterations, such as mutations, copy number variations, and epigenetic modifications, including changes in methylation[17].
DNA methylation is a key epigenetic modification characterized by the addition of a methyl group to the 5' position of cytosine rings within CpG dinucleotides, which are pairs of cytosine (C) and guanine (G) nucleotides linked by a phosphate group. This modification often results in the transcriptional silencing of associated genes[18]. In the context of cancer, aberrant DNA methylation—specifically, the hypermethylation of tumor suppressor genes and hypomethylation of oncogenes—plays crucial roles in tumor initiation and progression[19,20]. The detection of these methylation patterns in ctDNA has emerged as a promising noninvasive method for cancer diagnosis, prognosis, and monitoring. Because methylation changes occur early in tumorigenesis, ctDNA methylation analysis provides an opportunity for the early detection of malignancies, including EC. Consequently, DNA methylation profiling in ctDNA is becoming an integral component of personalized medicine, facilitating real-time monitoring of tumor dynamics and treatment response.
The detection of methylation biomarkers relies primarily on two technologies: (1) Polymerase chain reaction (PCR); and (2) Next-generation sequencing (NGS)[21]. PCR-based methods, such as methylation-specific PCR (MSP) and quantitative MSP (qMSP), are widely used for their specificity and sensitivity in detecting DNA methylation at specific loci[22]. These methods involve treating DNA with sodium bisulfite, which converts unmethylated cytosines to uracil while leaving methylated cytosines unchanged. MSP amplifies DNA based on these patterns, whereas qMSP quantifies methylation levels in real time, making it effective for monitoring tumor progression[23]. These techniques are particularly valuable for detecting methylation markers such as SHOX2, SEPTIN9, EPO, and RNF180 in ctDNA, providing a noninvasive diagnostic tool.
NGS also offers a more comprehensive approach by enabling high-throughput analysis of the genome or targeted regions, allowing for the simultaneous detection of multiple methylation sites[21]. Unlike PCR, which focuses on specific loci, NGS provides detailed methylation profiles that aid in identifying novel biomarkers and support precision medicine. In EC, NGS has been instrumental in revealing complex methylation patterns that drive tumor development, making it a powerful tool for early detection, diagnosis, and monitoring through methylation analysis.
The analysis of ctDNA methylation offers several advantages in the clinical setting. It enables early cancer detection, as the presence of specific mutations or methylation patterns can indicate tumor presence even before clinical symptoms arise. Additionally, ctDNA methylation can be utilized to assess minimal residual disease posttreatment, providing insights into the risk of recurrence[24]. This application of ctDNA methylation is particularly relevant in EC, where early diagnosis significantly impacts survival outcomes. Advances in technologies such as NGS and digital PCR have increased the sensitivity and specificity of ctDNA methylation detection, facilitating its integration into routine clinical practice.
KEY METHYLATION MARKERS FOR EARLY DETECTION
In the early detection of EC, methylation biomarkers present promising avenues for clinical diagnosis. Key markers such as SHOX2, SEPTIN9, EPO, and RNF180 have emerged as significant indicators of malignancy. Methylation of these genes plays a pivotal role in the molecular mechanisms driving cancer progression, including that of EC. DNA methylation typically occurs at CpG islands, which are regions of the genome that are rich in CpG dinucleotides and are often found in the promoter regions of these genes. Methylation at these sites leads to transcriptional silencing by preventing the binding of transcription factors and recruiting chromatin-modifying proteins. In the case of SHOX2, hypermethylation results in the loss of its regulatory roles in cell proliferation and differentiation, contributing to tumor growth[25-27]. Similarly, SEPTIN9, which regulates cell division, becomes silenced through methylation, disrupting normal cytokinesis and promoting uncontrolled cell proliferation[28]. EPO, which is primarily responsible for red blood cell production in response to hypoxia, undergoes hypermethylation, which silences its expression, allowing cancer cells to evade normal hypoxia-induced cell death and thereby supporting tumor aggressiveness[29]. Finally, RNF180, a tumor suppressor involved in protein degradation and cell cycle regulation, is silenced by methylation, preventing it from controlling cell growth and apoptosis, thus enabling unchecked tumor development[30]. These methylation-driven silencing events in ctDNA provide crucial biomarkers for the early detection, diagnosis, and monitoring of EC.
The study by Liu et al[9] evaluating the combined use of four methylation markers—SHOX2, SEPTIN9, EPO, and RNF180—represents a significant advancement in the early detection of EC. The combined analysis of these markers effectively distinguished esophageal adenocarcinoma from squamous cell carcinoma, EC, and controls without any tumors, with areas under the operating characteristic curve of 0.864 for EC, 0.737 for esophageal adenocarcinoma and 0.824 for SCC, significantly outperforming squamous cell carcinoma antigen, class antigen 199 (CA199), and carcinoembryonic antigen (CEA). By demonstrating that the simultaneous assessment of these markers can substantially improve diagnostic sensitivity (76.19%) and specificity (86.27%), this study underscores the potential of a multimarker approach to enhance early diagnosis. This combination allows for a more comprehensive understanding of the tumor's epigenetic landscape, which may facilitate the identification of malignant transformation at an earlier stage than currently possible with single markers. Moreover, the inclusion of 210 EC patients and 102 controls strengthens the statistical reliability of the findings and ensures broad applicability across clinical settings. By including patients in both early and advanced disease stages as well as healthy controls, the study's results are relevant for a wide range of patients. In particular, the focus on early-stage disease detection is critical given the current limitations in diagnostic tools for EC, adding significant clinical value. Additionally, the fact that the study employed a robust methodology to measure methylation markers in plasma samples increases the potential for these markers to be used in noninvasive liquid biopsy techniques, offering clinicians a reliable and accessible tool for monitoring at-risk populations. Overall, the findings of this study not only support the clinical utility of these combined methylation markers but also pave the way for further research into integrated biomarker panels for improved diagnostic accuracy in EC.
CHALLENGES AND FUTURE DIRECTIONS IN CURRENT PRACTICE
Liquid biopsy offers significant advantages over traditional diagnostic methods such as endoscopy by enabling safer, noninvasive blood draws that allow real-time tumor monitoring and capture of tumor heterogeneity[13,31]. This approach enhances the early detection of EC through the identification of biomarkers such as ctDNA and methylation patterns, which are often detectable before clinical symptoms appear. Moreover, ctDNA is also valuable in monitoring treatment efficacy and long-term management, as changes in ctDNA levels during and after treatment allow clinicians to assess therapy response, detect minimal residual disease, and predict recurrence[32]. This real-time monitoring enables more personalized treatment adjustments and long-term surveillance, ultimately improving the prognosis for patients with EC.
Although promising, current assays for detecting early-stage EC face limitations in sensitivity and variability in the specificity of methylation markers across different populations. The sensitivity and specificity may differ between early-stage and late-stage disease or across different EC subtypes, such as adenocarcinoma and squamous cell carcinoma, due to the distinct molecular profiles and tumor burdens at different stages. The potential influence of nontumor-derived ctDNA, which can be released from healthy or inflamed tissues, may also affect the accuracy of these biomarkers, leading to false positives. The lack of large-scale, multicenter validation studies is a critical limitation that needs to be addressed to confirm the reliability and consistency of these markers across various demographic groups, disease stages, and geographical regions. To ensure clinical relevance, it is essential to validate these biomarkers through large-scale studies involving diverse patient cohorts to account for population-specific differences. Additionally, there are significant challenges related to the cost and accessibility of these assays. The high cost of technologies such as NGS and PCR-based methods could limit their widespread use, particularly in resource-limited settings where such testing is not financially feasible. Moreover, accessibility remains an issue, as the infrastructure needed for liquid biopsy analysis is still predominantly available in specialized centers, making it less accessible to patients in rural or underserved areas. Future research should focus on integrating methylation markers with other biomarkers, utilizing machine learning and bioinformatics to develop composite scoring systems that increase diagnostic precision. For example, combining methylation markers with tumor markers such as CEA or CA199 or even imaging data could improve diagnostic accuracy. Machine learning techniques, such as decision trees or support vector machines, can be used to identify patterns in complex datasets, helping to create more accurate predictive models. Incorporating these markers into routine screening programs and clinical guidelines will require standardized protocols, clear criteria for interpretation, and integration into existing diagnostic frameworks. This comprehensive approach will help establish their role in early detection, facilitating more effective monitoring, personalized treatment, and improved patient outcomes in EC management.
CONCLUSION
The use of ctDNA and methylation biomarkers is revolutionizing the diagnosis and management of EC. Noninvasive liquid biopsy techniques enable early detection through the identification of key methylation markers, such as SHOX2, SEPTIN9, EPO, and RNF180, which increase diagnostic sensitivity and specificity. Despite existing challenges, further validation and integration of these biomarkers with other diagnostic modalities hold promise for advancing personalized medicine and improving patient outcomes in EC management. Additionally, ctDNA, beyond its key role in early diagnosis due to its minimally invasive nature, shows great promise in predicting recurrence and aiding postoperative follow-up, making it a valuable tool for clinicians and playing a crucial role in personalized therapy and precision medicine.
Provenance and peer review: Invited article; Externally peer reviewed.
Peer-review model: Single blind
Specialty type: Gastroenterology and hepatology
Country of origin: China
Peer-review report’s classification
Scientific Quality: Grade B, Grade C, Grade C
Novelty: Grade B, Grade C, Grade C
Creativity or Innovation: Grade B, Grade C, Grade C
Scientific Significance: Grade B, Grade B, Grade B
P-Reviewer: Pan D; Salimi M; Wan J S-Editor: Luo ML L-Editor: A P-Editor: Zhao S
