Role of circulating tumor DNA methylation in gastric cancer initiation and progression: A comprehensive review

Abstract

Circulating tumor DNA (ctDNA) is the free DNA released by tumor or circulating tumor cells, which is associated with many tumor characteristics and can be used as a biomarker for early screening, monitoring, prognosis, and prediction of therapeutic response in patients with cancer. The field of gastric cancer is very attractive because there are no high-quality screening, monitoring, or prediction methods. Gastric cancer is characterized by great tumor heterogeneity, great differences in genetic and epigenetic characteristics among different subgroups of gastric cancer, and high sensitivity and specificity of methylated ctDNA, which is conducive to the identification of tumor genotypes and the formulation of accurate diagnostic and treatment strategies. In addition, many studies have confirmed that methylated DNA has unique advantages in predicting treatment response, adjuvant therapy, and drug resistance and can be used to increase the efficacy of chemotherapy regimens, improve the chemotherapy response of patients in the future, and even treat multidrug resistance. However, methylated ctDNA also faces many problems, such as low sensitivity and specificity in a single target, limited association between some gastric cancer subtypes and ctDNA, risk of off-target effects, and lack of large-sample and high-quality clinical research evidence. This review mainly summarizes the current research on the DNA methylation of circulating gastric cancer tumors and links these findings with the early screening of gastric cancer, recurrence monitoring, and potential treatment opportunities. With the advancement of technology and the deepening of cross-research between doctors and professionals, ctDNA detection will reveal more disease information and become an important basis for the field of gastric cancer and precision medicine treatment.

Key Words: Gastric neoplasms; DNA methylation; Circulating tumor DNA; Biomarkers; Early screening

Core Tip: Circulating tumor DNA (ctDNA) has emerged as a promising noninvasive biomarker for early cancer detection and monitoring. Aberrant DNA methylation is one of the earliest and most frequent epigenetic alterations in gastric cancer. This review summarizes recent advances in understanding the role of ctDNA methylation in gastric cancer initiation and progression, and its potential clinical applications. We highlight the advantages of ctDNA methylation analysis in disease diagnosis, prognosis prediction, and therapeutic monitoring. A better understanding of ctDNA methylation dynamics may provide new insights into personalized medicine and improve outcomes in patients with gastric cancer.

INTRODUCTION

The emergence and progression of gastric cancer involve many genetic pathways and epigenetic alterations[1-4]. A significant alteration in gene regulation that contributes to gastric cancer is aberrant methylation, which manifests as excessive methylation in specific gene regions or a global loss of methylation throughout the genome[5-7]. During apoptosis or necrosis, nucleic acid fragments termed circulating free DNA (cfDNA) are released into the bloodstream. A portion of cfDNA originates from tumors and has tumor-specific molecular markers; thus, it is referred to as circulating tumor DNA (ctDNA)[8-12]. Numerous intrinsic characteristics of ctDNA, such as its DNA methylation state and histone methylation patterns, can serve as indicators for early cancer detection, monitoring recurrence, and assessing therapy efficacy. Recent research[13-15] has demonstrated that alterations in DNA methylation status occur in precancerous lesions, which can facilitate noninvasive early screening and detection of stomach cancer. Furthermore, alterations in methylation status offer substantial benefits in tracking postoperative recurrence and predicting patient prognosis[16-20]. Moreover, numerous studies have indicated that aberrant gene methylation is correlated with recurrence, metastasis, and treatment efficacy[21-24]. The methylation status of ctDNA can facilitate early tumor identification, monitor tumor growth and recurrence during treatment, and assist in guiding specific tumor therapies. For patients requiring adjuvant therapy, medication resistance is an unavoidable and significant issue[25-28]. Research indicates that DNA methylation is correlated with treatment resistance in gastric cancer. Nonetheless, it is reassuring because DNA methylation is a reversible phenomenon[29-33]. Consequently, reversing aberrant epigenetic alterations may provide a viable approach to overcome drug resistance. The clinical manifestations of early stomach cancer are subtle, and imaging studies have low sensitivity for early detection, complicating the diagnostic process. Research on the methylation state of ctDNA[34-36], both nationally and internationally, is progressing swiftly owing to advancements in the comprehension of epigenetic mechanisms[37-40]. Numerous studies[41-46] have established that aberrant DNA methylation significantly contributes to the development of gastric cancer and has substantial potential for early screening, monitoring of recurrence and metastasis, evaluating therapy response, and managing drug-resistant patients. In summary, recent advancements in research regarding ctDNA methylation status as a biomarker for gastric cancer have been reported, and its clinical applicability in early diagnosis, recurrence monitoring, and treatment response prediction has been assessed.

CTDNA IN GASTRIC CANCER

The type of tissue, degree of tumor differentiation, extent of dissemination, location of the tumor, and anticipated prognosis may all be associated with changes in the DNA methylation of critical genes in gastric cancer[47-50]. Furthermore, the methylation status of genes related to gastric cancer is influenced by environmental factors, including infection, chronic inflammation, diet, physical activity, age, and smoking[51-54]. Furthermore, the specific methylation status of gastric carcinoma exhibits a substantial age-related methylation pattern. This may be related to the vulnerability of elderly individuals to cancer[55-60].

The chronic inflammation caused by Helicobacter pylori (H. pylori) is one of the primary external factors that can influence gastric cancer because it may alter the methylation patterns of genes[61-65]. The methylation status of the cancer-related genes CDH1 and MGMT is entirely or partially reversed following H. pylori eradication; nevertheless, their DNA methylation levels do not recover to noninfected levels, which could lead to permanent gene imprinting. In contrast to H. pylori, which results in persistent inflammation and increased DNA methylation[66-70], Epstein-Barr virus (EBV) does not seem to cause an inflammatory phase, and its elevated DNA methylation is thought to be a direct consequence of infection. Blood or plasma sample-based liquid biopsies have demonstrated great promise in cancer research, particularly in the early identification and tracking of stomach cancer[71-76]. A noninvasive technique called liquid biopsy uses blood ctDNA analysis to provide vital information about cancers[77-80]. The occurrence and progression of malignancies are tightly linked to changes in the methylation state of ctDNA, and a small amount of DNA is released into the bloodstream by tumor cells (Figure 1).

Figure 1 Liquid biopsy performed by using blood/plasma samples. A: The bloodstream close to the tumor mass receives circulating tumor cells, extracellular vesicles, and free nucleic acids from the tumor; B: The main methods that have been used to analyze circulating free DNA in blood samples are real-time PCR, COLD-PCR, digital PCR, BEAMing, and next-generation sequencing. ctDNA: Circulating free DNA.

Internal influencing factors

The occurrence and progression of stomach cancer are facilitated by the epigenetic mechanism of cells, which frequently involves hundreds of active proteins, somatic mutations, and changes in the expression of epigenetic genome regulators[81-84]. Nonetheless, the relevant mechanism remains unclear, and the current study on the epigenetic mechanism of somatic mutations and epigenome regulators is insufficiently thorough[85-88]. One of the most prevalent mutations in ARID1A, a driver gene of gastric cancer, is frequently enriched in the EBV and microsatellite instability subtypes; nevertheless, how this mutation affects the overall chromatin or DNA methylation status of gastric cancer is unknown[89-93]. Gastric cancer has also been linked to changes in the transcription of genes encoding epigenetic enzymes, primarily as a result of decreased expression of the DNA demethylases TET1, DNMT1, and EZH2 and the histone methyltransferase SETDB2[94-98].

Relationship between ctDNA methylation and early diagnosis of gastric cancer

To increase the survival rate and quality of life of patients with stomach cancer, early detection and treatment are crucial[99-103]. To increase survival or lower incidence, the best early detection strategy is to find precancerous lesions or malignancies that may later emerge at the earliest stage of the disease as soon as possible[104-107]. The early detection of stomach cancer is aided by DNA methylation, which takes place during the early stages of tumor growth and can offer potential information for early diagnosis and treatment[108-110]. An aberrant DNA methylation status is not only a characteristic of advanced malignant tumors but also an early event and driving force in the pathogenesis of gastric cancer, according to recent studies. The methylation status of several genes varies in gastric cancer[111-115]. The DNA methylation status of genes linked to cancer was altered in the gastric mucosa of precancerous tissues, intestinal metaplasia tissues, and surrounding tissues in addition to actual gastric tumors. Inhibiting DNA methylation in animal models has also been shown by some researchers to lower the risk of stomach cancer[116-120]. Chronic inflammatory response-induced epigenetic alterations are believed to compound over time in H. pylori-positive gastric cancer, increasing the likelihood of cancer development[121-126].

There is either very little of these gene mutations in plasma or less mutant DNA released into the blood than what can be detected[127-130]. The high frequency of epigenetic aberrations in polyps and early gastric cancer, as well as the release of more methylated DNA into the blood, may be the reason why other studies[131-136] have demonstrated that changes in ctDNA methylation status are more common in these conditions than DNA mutations[137-140]. Therefore, we believe that finding methylated DNA could be more helpful for early screening of gastric cancer, even though the new detection technologies and methods being developed focus on improving the ability to detect DNA mutations (Figure 2).

Figure 2 Applications of circulating free DNA liquid biopsy in the recurrent and/or metastatic setting. A: Genotyping assays permit the deployment of precision-therapy umbrella enrichment studies that seek to identify specific actionable alterations and assign patients (randomly or not) to matched therapies of interest on the basis of a putative predictive biomarker; B: Dynamic and response-guided studies take advantage of the potential for changes in circulating free DNA (ctDNA) levels after treatment to anticipate clinical response to inform a change in therapy; C: To evaluate the utility of ctDNA genotyping and monitoring in the management of metastatic disease. ctDNA: Circulating free DNA. Created with BioRender.com (Supplementary material).

The pathological results of tissue biopsies are currently the gold standard for diagnosing stomach cancer; nevertheless, endoscopic biopsy has numerous drawbacks, and operating on large numbers of asymptomatic individuals can be challenging[141]. Additionally, various factors in laboratory tests and live studies affect common blood tests (such as CA72-4, CA19-9, and CEA) and stool tests for hidden blood, which also present problems in terms of accuracy and reliability. These markers have limited relevance in early-stage cancer screening, and even studies that combine numerous serological markers have demonstrated poor sensitivity[142]. However, these genes are strongly correlated with tumor stage and patient survival. It is therefore imperative to develop a simple, sensitive, and efficient detection method.

Identifying driver gene mutations in plasma cfDNA was the original strategy, and liquid biopsies are appealing for early cancer screening. However, in people without cancer, mutations related to clonal hematopoiesis of indeterminate potential that are not clearly important might affect this approach[143]. As technology has improved, we can now create products for clinical use by studying the differences in methylation gene patterns between the cfDNA of healthy individuals and that of patients with stomach cancer. SEPT9 has been used in clinical settings for more than a decade and is the first tumor biomarker authorized for the detection of colorectal cancer[144]. The amount of SEPT9 gene methylation was significantly greater in the blood of gastric cancer patients than in that of healthy controls, suggesting that this methylation in the serum could help in the early diagnosis of gastric cancer. The tumor suppressor gene RNF180 is highly methylated in gastric cancer, and its expression is markedly downregulated in primary gastric cancer[145]. It might be possible to use methylation of the promoter region, which is connected to a decrease in RNF180 gene expression, to screen for gastric cancer. It was discovered that SEPT9 methylation in blood had a sensitivity of 17.7% and specificity of 90.6% for detecting gastric cancer, indicating that this method can be helpful for early gastric cancer screening. However, the sensitivity was insufficient for clinical use. In a recent study on gastric cancer screening, the SEPT9 methylation marker alone had a sensitivity of 42.6%, whereas the use of CEA along with a protein marker increased the sensitivity to 86.4%, greatly improving the ability to diagnose the disease[146]. By combining detection with the protein marker CA72-4, their diagnostic capabilities could be further enhanced[147,148]. In summary, the combination of several markers and multitarget detection may be an effective early screening method for stomach cancer. The development of extremely sensitive detection techniques has made it possible to identify trace methylation alterations and low-frequency mutations, which are essential for early cancer detection and disease progression tracking. Currently, the two most popular techniques for detecting ctDNA are digital PCR (dPCR) and second-generation sequencing. dPCR can accurately identify small amounts of ctDNA molecules by using detailed measurements and very sensitive methods to separate them. Additionally, the use of bioinformatics along with machine learning and artificial intelligence (AI) can greatly improve the accuracy and reliability of detecting ctDNA (Figure 3).

Figure 3 Current approaches for the sensitive detection of circulating free DNA. MS: Mass spectrometry.

Role of DNA methylation in screening of gastric cancer

Stool is one of the most common analytes used in gastrointestinal screening tests. However, many challenges remain to be overcome in the early detection of gastric cancer via the DNA of shed cells[149]. The stomach is an upper digestive tract organ, and a large amount of gastric acid and a long gastrointestinal tract can damage the DNA of shed cells in the stomach. Some studies[150-152] have indicated that the detection of gastric cancer via DNA in feces may be ineffective.

Recent studies[153-155] have shown that the methylation status of the DNA of shed cells from gastric cancer patients can be detected from feces, and a diagnostic tool named "Colocaller" has been developed according to the methylation status of four targets; this tool has the potential to be used as an auxiliary diagnostic tool for gastric cancer. In addition, some scholars have reported that the promoter regions of TERT, RASSF2, and SFRP2 in the stool of patients with gastric cancer are hypermethylated. This also provides us with a reference method for exploring the diagnosis of gastric cancer using shedding cell DNA, that is, whether one or several "pancancer" markers can have a high screening effect on both gastric cancer and colorectal cancer. In the future, if fecal-based methylation detection has high sensitivity and specificity and retains simple and noninvasive characteristics similar to those of fecal occult blood tests, it will help popularize and improve gastric cancer screening and further improve the detection rate of early gastric cancer.

APPLICATION OF CTDNA METHYLATION IN MONITORING METASTASIS AND TREATING GASTRIC CANCER RECURRENCE

Application of ctDNA methylation in assessment of recurrence and metastasis of gastric cancer

For patients with lymph node-positive gastric cancer, the recurrence rate can still be as high as 88%, and the 5-year overall survival rate is only approximately 20%, even if they receive radical surgery and standard postoperative adjuvant chemotherapy. At present, there is a lack of means for monitoring gastric cancer recurrence, and the commonly used CEA and CA19-9 can monitor only approximately 40% of cases of tumor recurrence[156]. Key studies in gastric cancer have shown that ctDNA testing can identify patients with minimal residual disease (MRD) after surgery and assess patients' risk of postoperative recurrence. ctDNA has a short half-life, and if ctDNA is continuously detected in the blood after surgery, it may indicate the presence of tumor cells or micro-metastases in the blood circulation[157]. Therefore, the presence of postoperative ctDNA theoretically indicates the possible presence of MRD. Some studies have shown that the methylation status of ctDNA after surgery is an independent predictor of MRD, and patients with positive methylation are associated with an increased risk of MRD and recurrence. Another study revealed that CHFR, RUNX3, MGMT, and hMLH1 are highly methylated in primary gastric cancer and that these methylation genes help predict excessive tumor cells in regional lymph nodes. Machine learning also showed high accuracy in predicting the risk of gastric cancer lymph node metastasis via multitarget methylation analysis. This provides us with a new method and approach to predict early lymph node metastasis in the future, but high-quality studies on the relationship between methylated genes and early regional lymph node metastasis are lacking. Interestingly, MINT2 methylation levels in tumor tissues are very close to those in preoperative peritoneal lavage fluid (PPLF) and blood samples[158]. These findings suggest that abnormal MINT2 methylation in PPLF/blood may indicate the presence of peritoneal micro-metastases in patients with gastric cancer. If MINT2 methylation is positive before surgery, a variety of treatments, such as HIPEC therapy, may be used to reduce the possibility of postoperative recurrence and metastasis (Figure 4).

Figure 4 Schematic of BEAMing. ctDNA: Circulating free DNA; HP: High pH.

A 2020 study by Japanese researchers revealed that high levels of long-fragment LINE-1s after surgery might indicate MRD and a greater chance of cancer returning, whereas the amount of methylation in LINE-1s after surgery might not indicate MRD. Since a large amount of cfDNA comes from normal cells, the low methylation level of LINE-1 cfDNA indicates that many cancer cells with high methylation in the bloodstream might grow or spread easily. Additionally, the small number of gastric cancer patients suggests that there is uncertainty regarding the relationship between the degree of methylation and MRD[159].

The association between MRD in stomach cancer and ctDNA methylation status is still debatable, despite the paucity of research on this subject. However, existing research indicates that the ctDNA methylation status is a reliable indicator of MRD[160]. It can predict potential early lymph node metastases and peritoneal micro-metastases, determine the patient's risk of recurrence, and direct adjuvant therapy in the future. It can also reveal whether there are small residual lesions that are not visible on imaging examinations following surgery. While MRD patients can be found after standard follow-up treatment, which suggests that more tailored treatments, such as extra immunotherapy, targeted therapy, and personalized antigen vaccines, might be needed, the ctDNA methylation status can help identify high-risk patients for more aggressive treatment if it is found before follow-up therapy. The final stage of treatment ultimately erases the ctDNA methylation condition.

According to research on animals, stopping the aberrant methylation of DNA can prevent stomach cancer from developing. DNA methyltransferase inhibitors (DNMTis) are hence the subject of active pharmacological research. The use of DNMTis in conjunction with routine chemotherapy has been shown to correct aberrant gene regulation, enhancing the receptivity of cancer cells to treatment and decreasing their resistance to medications. In gastric cancer patients, for example, the combination of 5-fluorouracil and decitabine results in the over-methylation and sensitization of TFAP2E. A phase I clinical trial in which azacitidine was used to eliminate methyl groups in patients with advanced gastric cancer prior to neoadjuvant chemotherapy was conducted by researchers in the United States. They administered 5-azacitidine to gastric cancer patients prior to initiating neoadjuvant chemotherapy with the EOX (epirubicin, oxaliplatin, capecitabine) regimen. They discovered that some tumor-related genes, such as HPP1, TIMP3, CDKN2A, ESR1, and MGMT, had an excessive number of methyl groups attached, and preliminary findings showed that patients generally tolerated the neoadjuvant VEOX regimen. The effectiveness of chemotherapy may be increased by 5-azacitidine prechemotherapy. Of course, further neoadjuvant regimens and high-quality randomized studies are needed to further ascertain whether the combination of chemotherapy with neoadjuvant regimens is superior to chemotherapy alone. Adjuvant radiation therapy for stomach cancer has been controversial until recently, and it is unclear which patients may benefit from it. It is feasible to predict the impact of adjuvant radiation on the basis of the DNA methylation state of specific genes because studies[161-163] have demonstrated that radio-resistance in gastric cancer cells may result from the hypermethylation and inactivation of certain cancer-related genes. Furthermore, recent research has suggested that particular DNMTis can be utilized as radiation sensitizers to treat specific forms of gastric cancer and increase the radiosensitivity of gastric cancer cells[164]. DNMTis work in concert with radiotherapy and chemotherapy medications, and they can be utilized as sensitizers for both treatments. Therefore, in the future, DNMTs that target DNA methylation may be employed to cure and enhance multidrug resistance in gastric cancer. To prevent or reverse aberrant epigenetic changes early in disease treatment, the emerging therapeutic discipline of drug epigenetics focuses on important epigenetic targets. Clinical and preclinical trials[147,148,165] are currently being conducted to examine a growing number of medications that target DNMTis, such as the novel DNMT inhibitor zebularine. Histone methylation medications are still in their infancy as potential targeted therapy avenues; however, they are comparable to DNMTis. Additionally, research has shown that the CRISPR-Cas9 system is a promising targeted therapeutic approach for DNA methylation. In light of this, directed methylation editing might be more promising than DNMTis, which is more consistent with the idea of precision medicine.

Few prospective or clinical trials exist, and the majority of the studies that are now available are retrospective and small sample studies, which often lead to selection bias. Therefore, there is an urgent need for high-quality, large-scale, in-depth research (Figure 5).

Figure 5 Schematic diagram of the DNA-rN1-DNA-mediated SERS-based platform for circulating free DNA detection. ctDNA: Circulating free DNA; WES: Whole exome sequence; WGS: Whole genome sequence.

Early detection can greatly increase the survival rate and quality of life for patients with stomach cancer; nevertheless, patients with middle- and advanced-stage gastric cancer must weigh the relative benefits of various treatments. DNMTis can increase the efficacy of conventional chemotherapy regimens, address multidrug resistance, and predict treatment response, adjuvant therapy regimens, and drug resistance[166]. They also have special advantages in these areas. Furthermore, DNA methylation might be associated with the tumor microenvironment, which could predict how well adjuvant immunotherapy and even gastric cancer immunotherapy would work (Figure 6). However, many chromatin-modifying enzymes have more varied activities and do not constantly evolve in the way we anticipate, and these methylating medications and regulators still carry the potential for off-target harm. For future epigenetic therapies to be successful, comprehending the underlying mechanisms of DNA methylation and finding new targets and medications are crucial.

Figure 6 Development of a nanoparticle coupling tactic for detecting target KRAS mutations. ctDNA: Circulating free DNA.

CONCLUSION

To anticipate therapy response, track relapses, and conduct early illness screening, interdisciplinary collaboration is essential. The use of AI to help find and validate markers in the process of creating screening models, for example, could accelerate the process and even increase the precision of prediction models. However, few papers on DNA methylation-based biomarker AI models exist. A single biomarker may also have limited accuracy and specificity; future multimodal, multi-sample combination markers may be able to reach higher sensitivity and specificity. Without a doubt, epigenetics is a new area of study for gastric cancer. As more potent and affordable technologies and high-caliber clinical research become available, epigenetic indicators for gastric cancer may become more comprehensive and objective. To routinely use epigenetic markers for early identification, recurrence monitoring, and therapy effect prediction, more information is needed.