Accumulation of genetic and epigenetic changes ultimately lead to the initiation and progression of cancer (2,3). Genetic mutations have long been considered a major cause of cancer, but more recently, epigenetic changes have also been suggested to be important factors in cancer development (4). The unified definition of epigenetics remains ambiguous; however, more researchers support Holliday's definition as it offers two aspects of epigenetics, that is, ‘the study of the changes in gene expression, which occurs in organisms with differentiated cells, and the mitotic inheritance of given patterns of gene expression’ and ‘nuclear inheritance, which is not based on differences in DNA sequence’. The streamlined definition is ‘the study of changes in gene function that are mitotically and/or meiotically heritable and that do not entail a change in the DNA sequence’ (5).

The study of DNA forms the cornerstone of genetics, while epigenetics is the study of changes in gene expression that do not involve alterations in the underlying DNA sequence. Common epigenetic changes in different cancer types include abnormal DNA methylation, abnormal histone modifications and changes in the expression levels of numerous non-coding RNAs (6). Abnormal DNA methylation was the earliest identified modification and is the most widely studied epigenetic change (7).

For hypermethylated DNA to be a valid biomarker, it should be available via minimally invasive procedures and from tumor-remote media. Moreover, it must be effective in detecting the disease at an early stage. Based on these characteristics, research on cancer-specific hypermethylated genes has focused on tumor-derived cfDNA.

Cancer cells release nucleic acids, proteins, vesicles and other biological components into the blood and other body fluids (8). Among these potential biomarkers, tumor-derived cfDNA has been examined over the past decade as an important tool in oncology precision medicine. Abnormal DNA methylation is one of the characteristics of numerous cancer type, and most importantly, it can be detected in cfDNA in blood, urine, feces and other biological samples. Moreover, cfDNA methylation is used for early detection of cancer, minimal residual disease surveillance, prediction of treatment response and prognosis and tracing of tissue origin.

DNA methylation markers have numerous advantages (9). Firstly, DNA methylation occurs early in tumorigenesis and can be tissue- and cancer-type specific. Secondly, it is consistent across multiple genomic regions and can be detected using numerous CpG dinucleotides. Finally, and most importantly, methylation patterns are often associated with the origin of specific cancer type, and thus, can be used to reveal the tissue origin.

The landmark for cfDNA methylation analysis was the development of a screening test for CRC, based on the methylated septin 9 (mSEPT9), which was approved by the Food and Drug Administration (FDA) in 2016 (10). At the same time, accumulating evidence verified the value of cfDNA methylation as a biomarker for the diagnosis and evaluation of cancer.

Several methylation markers have been found to be associated with the initiation and progression of CRC (2,3,22). Some of these markers show potential for early detection (Table I), prognostic evaluation (Table II), and even prediction of treatment response to CRC. The status of methylation markers is associated with various conditions, such as tumor size, grade or metastasis (Fig. 1). Therefore, methylation markers, such as O⁶-methylguanine-DNA methyltransferase (MGMT) and long interspersed nucleotide element-1 (LINE-1) (23,24), may have a cross-over role in the prognostic assessment of CRC, prediction of treatment response and even early diagnosis, in different conditions. Thus, to achieve the best predictive effect, the examination of multiple methylation markers, multitarget stool DNA (MT-sDNA) and fecal occult blood testing should be combined.

The SEPT9 gene is a tumor-suppressor gene, which is closely associated with the development of tumors and other types of human diseases. SEPT9 is involved in a variety of important physiological processes, such as cytokinesis, DNA repair, cell migration and apoptosis (25). Abnormal methylation reduces the activity of SEPT9 gene transcription, leading to dysregulated gene expression and abnormal physical function, which may eventually lead to the development of cancer (26).

Toth et al (27) reported that the protein expression level of SEPT9 in CRC was significantly lower compared with that in normal epithelial cells, and that the mRNA expression level of SEPT9 was decreased during the transformation from adenoma to CRC. Moreover, Wasserkort et al (28) examined the methylation status of SEPT9 in different types of colon lesions and their adjacent tissue, and suggested that hypermethylation of SEPT9 may be a late event in the progression of adenomas to CRC. This may be the reason why the sensitivity of SEPT9 gene methylation detection in adenoma is lower compared with that of CRC. A recent meta-analysis revealed that plasma mSEPT9 was of high diagnostic value for CRC and was significantly correlated with CRC stage (29). In addition, survival analysis indicated that there was a negative correlation between SEPT9 methylation levels and disease-free survival (DFS) after CRC surgery (30). Song et al (31) also reported that an SEPT9 assay displayed 75.1% sensitivity and 95.1% specificity for CRC detection. Notably, the detection rate for CRC stage 0 was 57%, for stage I it was 64%, and for stage II it was 88%, which was valuable for population screening, alone or combined with other methods (31).

In a previous study that included 1,544 CRC samples (stages I–IV), a prospective evaluation of SEPT9 trial was retrospectively conducted. The sensitivity and specificity of CRC were reported at 68.2 and 78.8%, respectively, while the sensitivity for advanced adenoma was 21.6%. The main performance characteristics of mSEPT9 were demonstrated by this study (30), and mSEPT9 was eventually approved by the FDA for CRC screening (10).

The SDC2 protein is a membrane protein that serves a role in cell proliferation and migration, and helps maintain cell integrity. The SDC2 gene is not expressed in epithelial cells of normal colonic tissues, but is expressed in mesenchymal cells (32).

Important evidence has been discovered using different analyses of methylation conducted in comprehensive experiments. For example, CpG sites of SDC2 abnormal methylation have been observed in the tumor tissues of the majority of patients with CRC, thereby showing the significant potential of SDC2 methylation in the early detection of CRC. In addition, the level of SDC2 DNA methylation in feces is closely associated with the occurrence of CRC, but not with clinical stage (33,34). Other studies have reported that SDC2 methylation may be highly sensitive in the detection of both advanced adenoma and CRC. Oh et al (35) examined the methylation of the SDC2 gene in primary tumors, adenomatous polyps, hyperplastic polyps and normal tissues, and identified that the positive SDC2 methylation was 100, 90.6, 94.1 and 0%, respectively. SDC2 methylation was also significantly elevated depending on the severity of the lesion. The overall sensitivity for CRC and small polyps was 90.0 and 33.3%, respectively, and the specificity was 90.9% for SDC2 methylation in fecal DNA. Some previous studies indicated that the diagnostic efficacy of SDC2 methylation had a sensitivity of 77.4–81.1% and a specificity of 88.2–98% in fecal DNA for CRC (36,37). Thus, SDC2 methylation may be a promising non-invasive biomarker for the early detection of CRC. It has also been reported that the combination of multiple biomarkers may be an effective strategy for improving the sensitivity and specificity of early cancer diagnosis (38).

MGMT, which encodes five exons and four introns, is located at 10q26 on chromosome 10 and the MGMT protein acts as a DNA repair enzyme (16). Abnormal hypermethylation of the MGMT promoter is associated with a lack of mRNA expression, accompanied by the loss of protein content and enzyme activity (16,23). In total, ~40% of metastatic CRCs exhibit silencing of the MGMT gene, which results in a corresponding inhibition of protein synthesis (39). Previous studies have reported that MGMT promoter methylation was the main cause of MGMT gene expression disorder (40).

Shima et al (40) evaluated 855 cases of stage I–IV CRC for MGMT using two methods, methylation-specific PCR and immunohistochemistry. The results demonstrated that the methylation rate was 38% and the loss of MGMT expression was 37%, and the consistency of the two methods was 81%. Sartore-Bianchi et al (41) screened the molecular characteristics of 2,044 patients with mCRC and then found that the MGMT promoter hypermethylation proportion was 48.7%. In another study that included 70 patients with CRC, MGMT hypermethylation was detected in serum-free circulating DNA, and MGMT promoter hypermethylation was observed in 90% of CRC cases, while no MGMT hypermethylation was found in the serum of healthy subjects (23). Freitas et al (42) reported that a combination of methylated MGMT and SEPT9 present a 93.8% sensitivity, 82.0% specificity, respectively, for CRC detection in tissue samples. Therefore, it was suggested that MGMT may be used as a clinical biomarker for early diagnosis of CRC. However, there are some differences in the positive rate of MGMT methylation in the aforementioned studies and, thus, future studies are required to verify the findings.

NDRG4, is involved in a variety of biological activities, such as cell proliferation, differentiation, development and stress. The protein sequences encoded by this family have a 52–65% homology and are highly conserved in the evolution of various species (43), with α/β hydrolase folding. Previously, researchers have identified NDRG4 promoter CpG island methylation as a potential and one of the most accurate marker for CRC detection (44), and this has been verified by an independent study (45).

Bagheri et al (46) revealed that the sensitivity and specificity of the NDRG4 gene in the diagnosis of CRC were 86 and 92%, respectively, and the proportion of methylation was proportional to CRC staging. Rademakers et al (47) combined types of biomarkers to establish a panel for the highest diagnostic potential, resulting in the combination of SNAP91/NDRG4/FIT as the best performing, with a sensitivity and specificity of 86.0 and 96.0%, respectively. Moreover, these authors demonstrated that the sensitivity and specificity of the NDRG4 gene were sufficient to serve as a novel, non-invasive marker for CRC screening. A meta-analysis also showed that NDRG4 could be considered as an important marker for the diagnosis of CRC (48).

In a previous study, the positive rates of methylated NDRG4 in cancer tissues, precancerous tissues, blood, urine and feces were shown to be 81, 8.3, 54.8, 72.6 and 76.2%, respectively. It was also confirmed that methylated NDRG4 in the feces and urine had high sensitivity and specificity. Additionally, this new method is expected to be a promising and potential marker for the early diagnosis of CRC due to the ease of collection of urine or feces samples (49,50).

APC is a tumor-suppressor gene that was discovered during the research of familial adenomatous polyposis. Furthermore, hypermethylation of APC promoters has been frequently observed in sporadic and familial CRC types over the past two decades (51).

A previous study of patients with CRC examined the methylation status of APC and found that APC methylation occurred 33% of the time in patients with CRC (52). And recent research also reported that in CRC patient under 50 years of age, the rate of APC promoter methylation was 40% (53). Nunes et al reported the methylation levels of APC combined with FOXA1 and RASSF1A to diagnose CRC in stages 0, I and II with 78.4% sensitivity, 69.9% specificity, respectively, but the result needs further validation because the CRC samples were only 37 (54). Moreover, methylated APC promoters were found to be significantly associated with later stages and an older age. The incidence of APC methylation in patients with mCRC was 53.7%. That study demonstrated that APC promoter hypermethylation is a common epigenetic event in patients with both early and metastatic CRC, and it serves an important role in the development of CRC (52). A meta-analysis by Liang et al (55) investigated APC hypermethylation in the early diagnosis of CRC. This study included 24 articles and 2,025 patients with CRC. The analysis demonstrated that APC promoter hypermethylation was an early cancerous event of CRC and, thus, may be a noteworthy diagnostic indicator of early CRC.

The first important evidence of BMP3 inactivation in early polyp formation and colon tumor development was reported in 2008 by Loh et al (56). These authors reported that the percentage of aberrant BMP3 hypermethylation in colorectal tumors was 55% (33/60). This result suggested that BMP3 may be a potential marker for the early detection of neoplastic lesions. In subsequent studies, researchers have focused on the diagnostic value, such as the sensitivity and specificity, of methylated BMP3 in CRC. For example, Houshmand et al (57) examined the methylation status of the BMP3 gene in CRC tissue samples, and detected a sensitivity of 56.66% and specificity of 93.3%. In addition, Rokni et al (58) detected the methylation status of the BMP3 gene in plasma DNA samples from 50 patients with histologically diagnosed polyps or tumors and 50 healthy individuals. Their results showed that the frequency of BMP3 methylated DNA was significantly higher in patients with polyps compared with that in healthy controls, with a sensitivity of 40% and specificity of 94%. In a study by Kisiel et al (59), the combination of abnormal methylated DNA markers with BMP3 and NDRG4 had a sensitivity of 100% and a specificity of 89% for CRC and high-grade dysplasia. These findings suggest that methylated BMP3 may be a useful biomarker for CRC detection, but that it should be combined with other biomarkers due to the lack of specificity. A typical example of a combination method is the application of MT-sDNA test, which consists of fecal hemoglobin, quantitative detection of methylation BMP3 and NDRG4, β mutant KRAS and β-actin tests. The sensitivity for curable stage CRC ranges from 93 to 100%, while its specificity ranges from 87 to 93%. Due to this reliable performance, it was approved by the US FDA (60).

VIM encodes a cytoskeletal protein that is considered to be involved in cancer invasion and metastasis. VIM promoter methylation has been observed in CRC (61), such as in the fecal DNA of patients with CRC (62). VIM promoter methylation has been detected in stool at a sensitivity of 52% and specificity of 88% (61). Lu et al (49) reported that the promoter methylation levels of VIM in patients with CRC were 41.1% and the specificity was 85.0%. These results suggest that promoter methylated VIM in fecal samples was sensitive, specific and non-invasive for CRC screening and, therefore, it has been commercialized as a non-invasive method for early CRC detection (63).

In addition, previous studies have suggested that the hypermethylation of the following genes, including ALX homeobox 4 (ALX4), neurogenin 1 (NeuroG1), helicase like transcription factor (HLTF), hyperpigmentation progressive 1 (HPP1), WNT inhibitory factor 1 (WIF1), Ras association domain family member 2a (RASSF2a), GATA binding protein 4 (GATA4), β-1,4-glucuronytransferase 1 (B4GAT1), proline rich membrane anchor 1 (PRIMA1), APC, ATM serine/threonine kinase, glutathione S-transferase π 1 (GSTP1) and tissue factor pathway inhibitor 2(TFPI2) (63,64), have been analyzed in fecal, blood and urine samples from patients with CRC and used for the early detection of CRC. From the potential methylation markers, methylated VIM, SEPT9, BMP3, SDC2, NDRG4 and a combination of methylated branched chain amino acid transaminase 1 (BCAT1)/IKAROS family zinc finger 1 (IKZF1) have been proven to be reliable and accurate, and have been approved for clinical use (10,60,63,65).

As shown by a recent meta-analysis (66), the value of a single hypermethylated DNA promoter region as a marker for CRC screening is limited, but the combination of indicators demonstrates significant potential. Therefore, the combination of different detection indicators must be utilized to balance their respective diagnostic performance, thus achieving the best diagnostic effect with regards to specificity and sensitivity.

Researchers have observed abnormal methylation of SFRPs in various types of human cancer. The activation of the Wnt pathway induced by the loss of SFRP gene expression is one of the most important mechanisms for tumorigenesis and cancer development (67). In humans, there are five types of SFRPs (SFRP1, 2, 3, 4 and 5). Among the SFRP family members, SFRP1 and SFRP2 have been the most extensively researched in human cancer, and studies have shown that SFRP1 and SFRP2 promoter methylation may contribute to the risk of CRC (68).

The loss of SFRP expression induced by DNA methylation is the primary mechanism causing the silencing of SFRP and it is associated with tumor formation in CRC (69,70). Kumar et al (71) evaluated the promoter methylation status of the SFRP1 gene in 54 cases with stage II–III CRC. It was found that SFRP1 gene methylation was associated with lymph node invasion (P=0.05) and a poorer mean overall survival (OS). Moreover, these authors indicated that SFRP1 gene methylation was a prognostic marker in CRC (71). It has also been shown that the hypermethylation rate of the SFRP2 promoter in patients with CRC was 66.7% (72). In addition, a recent meta-analysis of the SFRP family revealed a significant association between hypermethylation of SFRP1 and cancer risk (67). A review by van Loom et al (73) revealed that numerous studies have reported promotor hypermethylation downregulated SFRP2 gene expression in several types of cancer. These authors discussed the role of SFRP2 in tumor angiogenesis and noncanonical Wnt signaling, and suggested its potential as an anti-angiogenic therapeutic target or an effective prognostic marker in cancer.

p16 is a tumor-suppressor gene frequently studied due to its significant function in the cell cycle. There are three main mechanisms of p16 inactivation: Gene mutation, homozygous deletion and 5′-CpG island methylation, which is one of the major causative factors of various human cancers (74).

Ye et al (75) detected p16 methylation in CRC tissues and adjacent normal tissues, and the results demonstrated that p16 methylation was higher in CRC tissues compared with adjacent normal tissues. Moreover, Lee et al (76) found that hypermethylation of the p16 gene was significantly associated with lymph node metastasis in CRC, and reported that the hypermethylation frequency of the p16 gene was 32.3%. However, p16 gene promoter hypermethylation frequency was detected at 15.1% in CRC, and this rate was lower than that previously determined (72). This variation may be caused by the different sensitivity of detection methods, which can be further verified in future studies. Karam et al (74) reported a sensitivity of 55.38%, specificity of 98.5% and diagnostic accuracy of 77.7% in patients with CRC. Furthermore, p16 methylation was significantly correlated with age, sex, Dukes'stage, lymph node metastasis, carcinoembryonic antigen levels, a shortened time to progression and overall survival (75,77). Therefore, it may be associated with prognosis and may thus serve as a prognostic biomarker.

LINEs are retrotransposable elements identified in numerous eukaryotic genomes. These are highly methylated in normal somatic cells and, therefore, are mostly inhibited, thus preventing their potential to cause genomic instability (78). Full-length LINE-1 can lead to adenoma formation and cancer progression, and the activity of LINE-1 is dependent on the epigenetic regulation of its promoter (79). Hypomethylation of the LINE-1 promoter has been observed in gastrointestinal cancer as well as other types of cancer (79,80).

Jiang et al (81) examined promoter methylation of LINE-1 in adenomas and sessile serrated lesions (SSLS); the results showed that high-grade dysplasia (HGD) and increasing size of adenoma were associated with decreased LINE-1 methylation. Shademan et al (78) quantified promoter methylation and LINE-1 transcripts in three stages of CRC, non-advanced adenoma, advanced adenoma and adenocarcinoma. Their results demonstrated that the methylation of the LINE-1 promoter in non-advanced adenoma was significantly higher compared with that in advanced adenoma and adenocarcinoma. The correlation analysis also revealed a decrease in LINE-1 promoter methylation, genomic polymorphism insertion and an increase in LINE-1 transcription in advanced adenomas, suggesting that early and late polyps may involve some main pathogenetic mechanisms that ultimately lead to cancer.

Bachitta et al (82) conducted a meta-analysis in 2014, which described the role of LINE-1 hypomethylation in human cancer. It has also been shown that LINE-1 hypomethylation in CRC was associated with poor prognosis, survival and advanced stage, and that it could be a prognosis predictor of CRC (83,84). In addition to its prognosis value, a recent study reported that LINE-1 hypomethylation was effective in the early detection of CRC (85). Hence, LINE-1 hypomethylation may be a promising marker for the prognostic and early detection of CRC.

BCAT1 controls the metabolism of branched-chain amino acids, which are essential nutrients for growth. Abnormal methylation of the BCAT1 gene locus has been observed in CRC and various pathologic conditions (86). IKZF1 encodes a DNA-binding protein, which regulates the cell cycle. In CRC, IKZF1 promoter methylation is associated with the loss of regulation of cell proliferation and differentiation (87).

Mitchell et al (88) demonstrated that the expression levels of the two genes, BCAT1 and IKZF1, were low in healthy human plasma (3.5 and 4.9%, respectively) but were significantly increased in patients with CRC; therefore, these two genes were selected as biomarkers for CRC detection. In a previous study that included 2,127 samples, 85/129 cases of CRC had a sensitivity of 66%. For stages I, II, III and IV, the positive rates were 38, 69, 73 and 94%, respectively. It was also found that the positive rate was positively correlated with the degree of invasion (89). In a trial involving 1,381 volunteers, which examined FIT and BCAT1/IKZF1 DNA methylation, the results demonstrated that the sensitivity of the BCAT1/IKZF1 methylation to CRC was 62%, with a specificity of 92%, which was higher compared with the commonly used positive threshold of FIT. When combining FIT (cut-off 10 µg Hb/g) with the BCAT1/IKZF1 blood test, the sensitivity for cancer was 89%, with an improved specificity of 74% (90). From research on recurrent CRC, postoperative patients who were BCAT1/IKZF1-positive were found to have an increased risk of residual disease and subsequent recurrence. It has been shown that the BCAT1/IKZF1 test was more sensitive to recurrence compared with the conventional CEA test, and in the case of positive test, the recurrence rate was twice as high as that of CEA (91,92). The BCAT1/IKZF1 test is a novel blood test that have been approved under the commercial name ‘COLVERA’ for CRC (65), and could be used for monitoring recurrence and as a non-invasive diagnostic method.

RASSF1A is a tumor-suppressor gene that serves a role in regulating cell proliferation and apoptosis, and is involved in numerous types of cancer, including CRC. More importantly, it can be detected early in liquid biopsies (93). RASSF1A is subject to epigenetic regulation and suppression, the main regulatory mechanism being via DNA methylation (94). It has been reported that the development of adenomatous polyps and colorectal laterally spreading tumors (LSTs) are precursor lesions of CRC, and 80–90% of CRCs develop via these factors (95). In a previous study, Ni et al (96) detected the degree of RASSF1A methylation in patients with LSTs. The results indicated that RASSF1A methylation was lower in patients with LSTs compared with CRC cases, but was higher than that in polypoid adenomas. Thus, it was suggested that RASSF1A has the potential to be used for the early diagnosis of CRC and may enable timely intervention.

Promoter methylation of RASSF1A can affect the sensitivity of patients with CRC to oxaliplatin-based chemotherapy. In a study by Sun et al (97), it was identified that RASSF1A methylation was independently correlated with the prognosis of patients treated with oxaliplatin-based chemotherapy, while another meta-analysis revealed that RASSF1A hypermethylation was a risk factor and a potential prognostic biomarker in CRC (98). Moreover, it was indicated that the methylation of RASSF1A may be a prognostic marker for stage II and III CRC cases. In addition, with further research on the mechanism of methylation, aberrant methylation may be a promising and novel target to improve chemotherapy efficacy.

hMLH1 is considered to be an important member of the mismatch repair gene, and it encodes a variety of DNA repair enzymes to cooperate in the recognition and repair of DNA mismatches (99). Abnormal hypermethylation of the hMLH1 promoter CpG island has been shown to be closely associated with the occurrence of CRC, and its epigenetic changes may affect DNA stability (100). In total, ~15% of CRC cases exhibit high levels of MSI, reflecting the dysfunction of the post-replication DNA mismatch repair system, which mainly occurs via HMLH1 gene silencing mediated by CpG methylation (101). It has been reported that a MLH1 promoter hypermethylation assay is a cost-effective method for identifying microsatellite-high (MSI-H) in patients with sporadic CRC (102).

Fu et al (103) evaluated 115 patients with stage II CRC who were assigned to four groups: CIMP⁺/MLH1-unmethylated, CIMP⁺/MLH1-methylated, CIMP⁻/MLH1-unmethylated or CIMP⁻/MLH1-methylated. The results demonstrated that the CIMP⁺/MLH1-unmethylated group was associated with higher aggressiveness and poorer prognosis. DFS and OS were predicted by CIMP/MLH1 methylation status, with the shortest DFS and OS observed in the unmethylated CIMP+/MLH1 group. This study revealed that the CIMP combined with MLH1 methylation status was meaningful for tumor subtype classification in patients with stage II CRC. In 2018, a meta-analysis, which that included 47 studies with 4,296 cases and 2,827 controls, suggested that hMLH1 methylation was associated with an increase in CRC risk (99).

Furthermore, Kuan et al (104) demonstrated that patients with CRC at an advanced stage who exhibited hMLH1 methylation in tumor tissue had a higher risk of CRC recurrence compared with patients with local invasion who had an unmethylated status. It has also been reported that the methylation of hMLH1 has significant potential in predicting treatment response, such as that to 5-FU-based chemotherapy, and survival (105,106). It was also observed in an in vitro study that hMLH1 might play a key role in cancer stem cells response to 5-FU (107).

Wnt signaling is a term used to describe a group of signaling pathways that regulate processes essential to physiological functions, including cell proliferation and differentiation. It is also closely associated with CRC (108). Wnt5a/b expression is associated with epithelial-to-mesenchymal transformation (EMT), and an increase in its gene expression has been observed in numerous malignancies, including CRC (109). EMT is a process that enhances the migration of epithelial cells (110). In cancer, EMT increases the ability of cancer cells to escape from the primary tumor, allowing them to infiltrate into neighboring tissues.

In most patients with CRC, genes that regulate the Wnt pathway display genomic and epigenomic abnormalities. The decrease of Wnt pathway inhibitor protein can facilitate the growth and metastasis of CRC, which is an important factor in determining patient prognosis (111). Wnt5A is one of the negative regulators of the Wnt pathway and is often inhibited by promoter methylation in CRC (112). Hibi et al (113) reported that the Wnt5A gene was detected in 35% of primary CRC cases and that it was methylated from the early stages of CRC. Furthermore, Kim et al (112) collected tissues from 194 patients with metastatic or recurrent CRC and detected the methylation status of Wnt5A. The results indicated that the methylation frequency of Wnt5A was 32.0%, which was similar to those of a previous study (113). Jiang et al (114) also found that, in 5-FU-treated CRC cases, the Wnt5A methylation status was significantly associated with longer progression-free survival and improved drug response, suggesting that 5-FU was more effective in CRC cases with Wnt5A hypermethylation. Therefore, the Wnt5A methylation status may predict the 5-FU treatment response in patients with CRC.