Detection of methylated SEPT9 DNA in peripheral blood for diagnosis of colorectal cancer

Abstract

Background

Colorectal cancer (CRC) remains a leading cause of cancer-related death worldwide. Although colonoscopy is effective screening method, its widespread adoption is hampered by poor compliance. Development of patient-friendly screening methods that are minimally invasive is of paramount importance to improve CRC screening participation.

Methods

A single-center case-control study was conducted. Blood samples were collected from 119 participants (79 CRC patients, 20 patients with colorectal adenomas, and 20 healthy colonoscopy-negative controls) during May 2023 and March 2025. The methylated SEPT9 (mSEPT9) DNA in peripheral blood was analyzed by droplet digital polymerase chain reaction (ddPCR).

Results

The sensitivity and specificity of mSEPT9 detection in peripheral blood for diagnosis of all stages of CRC patients was 68.35% and 95.00%, respectively. The positive rate were similar between left-sided CRC and right-sided CRC (65.31% vs 72.41%，p ＞ 0.05). mSEPT9 detection in all healthy people with normal colonoscopy were negative. When mSEPT9 detection was combined with CEA and CA199, positive rate could be raised to 82.67%.

Conclusions

Blood-based mSEPT9 detection by ddPCR method is a sensitive and specific method for non-invasive diagnosis of CRC. Combination with CEA and CA19-9 could improve its performance. It may serve as a viable alternative for individuals unwilling or unable to undergo invasive procedures.

Keywords

SEPT9 methylation colorectal cancer droplet digital PCR diagnosis

Introduction

Colorectal cancer (CRC) is one of the most common malignancies in the world. It is currently the third most common cancer and the second leading cause of cancer-related death worldwide.^1–2 The disease burden of CRC is expected to increase to 3.2 million new cases and 1.6 million deaths by 2040.³ The number of incident cases and deaths of CRC in China has been increasing in the past 30 years and will continue to increase in the next 25 years.⁴

CRC screening is an effective strategy to reduce disease mortality and improve the prognosis of patients through early detection and removal of precancerous lesions and early-stage cancers.⁵ High-quality screening methods are urgently needed to facilitate the early diagnosis of CRC. At present, traditional methods for CRC screening have certain limitations, such as low sensitivity or specificity, invasiveness or high cost, requirement for bowel preparation, which limit their application in clinical practice.⁶ Consequently, screening of CRC in general population according to the current guidelines remains suboptimal,⁷ creating a significant public health gap and leaving a substantial portion of patients undiagnosed until advanced stages.

Therefore, development of patient-friendly screening methods that are minimally invasive with high sensitivity and specificity is of paramount importance to fill the gap. Detection of tumor-derived, methylated DNA in circulating cell-free DNA (cfDNA) extracted from peripheral blood has emerged as a highly promising approach. Among various biomarkers, methylation of SEPT9 gene (mSEPT9) promoter has shown particular promise, as it is a frequent and cancer-specific epigenetic alteration in CRC.^8–12 Although previous studies have already reported on mSEPT9, its definitive role in real-world clinical practice, particularly for identifying colorectal adenomas and early-stage CRC, requires further concrete evidence.

Recent years, droplet digital polymerase chain reaction (ddPCR) has gained widespread attention because of improved sensitivity and specificity in detecting gene mutations, particularly for circulating tumor DNA in low concentrations.¹³ It is highly robust for analyzing low-abundance circulating tumor DNA in blood samples and has a lower requirement for sample size, making it ideal for non-invasive cancer screening. However, there are few studies reported on the ddPCR method for detecting mSEPT9 in CRC.

Therefore, this study was specifically designed to address this unmet need and evaluation the performance of a peripheral blood-based SEPT9 methylation assay in patients with different stages of CRC and colorectal adenomas in a single-center setting. The latest ddPCR method was used to achieve quantitative results.

Material and method

Research object

This study enrolled patients who were admitted to The Fourth Affiliated Hospital of Soochow University (Suzhou Dushu Lake Hospital) from May 2023 to March 2025. Inclusion criteria of CRC patients: (1) age＞18 years old (2) CRC confirmed by colonoscopy or other diagnostic methods with primary tumor (3) had not received previous CRC surgery, chemotherapy, or radiotherapy. Exclusion criteria: (1) combined with other primary malignant tumors (2) lack of important clinical data. Staging of CRC was performed according to the American Joint Committee on Cancer/Union for International Cancer Control (AJCC/UICC) 8th edition TNM staging system. 20 healthy people who had normal results of colonoscopy and 20 patients who underwent endoscopic treatment for colorectal polyps and pathologically confirmed as colorectal adenoma were selected as controls.

Blood samples of CRC patients were collected before surgery or chemotherapy. For healthy people and patients with colorectal adenoma, it was collected before colonoscopy. Samples were pretreated within 72 h after collection, and pretreated supernatant samples were stored at −80°C until DNA isolation. Clinical information of patients was blinded to examiners.

The study design adhered to the ethical principles outlined in the Declaration of Helsinki and was approved by the ethics committee of The Fourth Affiliated Hospital of Soochow University (Suzhou Dushu Lake Hospital) (approval number 220153). Written informed consent was obtained from all participants in this study.

mSEPT9 detection method

DNA isolation and bisulfite modification

DNA in plasma supernatant and urine supernatant were separated and purified using QIAamp Circulating Nucleic Acid Kit (QIAGEN), respectively. Separated DNA was eluted in 40μL of elution buffer. DNA concentration was measured using the Qubit™ dsDNA HS Assay (Invitrogen) kit. Sulfite treatment of DNA was performed using EZ DNA Methylation Gold kit (Zymo Research), and modified DNA was temporarily stored at −20°C. All procedures were performed according to the standard operating procedure of the kit manufacturer.

Droplet digital PCR (ddPCR)

Detection and analysis were performed with the use of a QX200™ Droplet Digital™ PCR System (Bio-Rad). A specific fragment in the promoter of the SEPT9 gene (RefSeq Gene ID: NG_011683.2) was targeted. S9-Probe1-M was a specific detection probe for methylated Septin9 sequence. S9-Probe1-U was a specific detection probe for unmethylated Septin9 sequence. The upstream and downstream primers were designed in regions lacking CpG dinucleotides. Specific information of the primers and probe sequences was shown in Table 1. Droplets were generated through a QX200 droplet generator (Bio-Rad) using 20μl ddPCR mixture and 70μl droplet generating oil (Bio-Rad). Samples were transferred to a 96-well PCR plate (Bio-Rad) and sealed. PCR was performed in a T100(Bio-Rad) thermal cycler with conditions set at 95°C for 10 min, 40 cycles of 94°C for 30sec and 57°C for 1 min, 98°C for 10 min; 4°C for 5 min. Fluorescence signals were detected by QX200 Droplet Reader (Bio-Rad). For each experiment, following control samples were included: a methylation positive control (commercially available in vitro methylated DNA; Thermo Fisher), a methylation negative control (bisulfite treated DNA from healthy human blood leukocytes), and a nontemplate control (nuclease-free ultrapure water; Gbico). All analyses were performed according to the digital MIQE guidelines.¹⁴

Table 1.

Specific information of the primers and probe sequences.

	Abbreviation	Sequence
Upstream primer	S9-F1	5′-GYGATTYGTTGTTTATTAGTTATTATG-3′
Downstream primer	S9-R1	5′-CCAACCCAACACCCACCTT-3′
Specific detection probe for methylation Septin9 sequence	S9-Probe1-M	5′-FAM-TCGGATTTCGCGGTTAA-MGB-3′
Specific detection probe for unmethylated Septin9 sequence	S9-Probe1-U	5′-VIC-TTGGATTTTGTGGTTAATG-MGB-3′

mSEPT9 detection data analysis

QX Manager Standard Edition software version 1.1 (Bio-Rad) was used for sample methylation data analysis. In 2D scatter plot, it was judged as positive if upper left quadrant positive droplet number is ≥2, and signal value > 4000, and number of positive signal microdroplets in the right lower quadrant was ≥2, and the signal value was > 4000. Gray dots indicate negative (empty droplets, no nucleic acid), whereas blue dots indicate positive methylation and green dots indicate positive unmethylation. Methylated and unmethylated SEPT9 copies in 20μL reaction and corresponding concentration (copies per μL) were calculated according to the proportion of positive droplets. Methylation abundance was calculated as the fraction of methylation concentration/total number of copies. Mutation frequency was calculated as the number of methylated droplets /(number of methylated droplets + number of unmethylated droplets)*100%. The mSEPT9 detection was defined as positive when number of positive methylation droplets was ≥2 and the mutation frequency was > 0.05%.

CEA and CA199 measurement

CEA and CA199 levels were measured by enzyme-linked immunoassay in the department of laboratory of the hospital. Abnormal CEA and CA19-9 level was define as above 5 ng/ml and 30 U/ml, respectively.

Data analysis

Statistical analysis was performed by the SPSS (version 23) and GraphPad Prism 10.3.1. Categorical data were compared by using the Chi-square test or Fisher's exact test. All statistical analyses were two-sided, and P < 0.05 was considered statistically significant difference.

Result

Enrollment and grouping

124 participants were enrolled in this study. 5 were excluded because lack of pathological diagnosis. 119 participants were included for analysis and grouped according to results of colonoscopy and pathology (Figure 1).

Figure 1.

Enrollment and grouping of participants.

Characteristics of all participants and CRC patients

Of 119 patients included, 79 had CRC, 20 had colorectal adenoma and 20 had normal colonoscopy. Clinical characteristic of all these participants and CRC patients was shown in Table 2 and Table 3. Average age of CRC patients was over 60 years old and more than half of them were male. Left-sided CRC were more common than righted sided CRC.

Table 2.

Characteristics of all participants.

	Healthy control (n = 20)	Colorectal adenoma (n = 20)	CRC (n = 79)
Male: Female	7:13	13:7	46:33
Age	50.7 ± 8.0	56.5 ± 12.8	65.6 ± 12.6
Hypertension	4 (20.00%)	7 (35.00%)	30 (37.97%)
Diabetes	4 (20.00%)	2 (10.00%)	10 (12.66%)
History of cholecystectomy	2 (10.00%)	0	6 (7.59%)
White blood cell count (10⁹/L)	5.41 ± 1.91	5.23 ± 1.14	6.45 ± 2.51
Hemoglobin (g/L)	140 ± 17	134 ± 16	115 ± 27
Platelet count (10⁹/L)	209 ± 43	221 ± 62	251 ± 107
Total bilirubin (μmol/L)	9.9 ± 3.9	11.6 ± 9.7	10.8 ± 6.4
Creatinine (μmol/L)	65.9 ± 12.2	92.9 ± 101.7	82.0 ± 84.8
Uric acid (μmol/L)	320.1 ± 84.9	305.6 ± 83.2	295.4 ± 102.3

Table 3.

Characteristics of CRC patients.

Stage of CRC cases(n = 79)
Stage I	20 (25.32%)
Stage II	18 (22.78%)
Stage III	25 (31.65%)
Stage IV	16 (20.25%)
Location of lesions(n = 78)*
Left-sided colon	49 (62.82%)
Right-sided colon	29 (37.18%)
Site of metastasis(n = 16)
Liver	10 (62.50%)
Lung	2 (12.50%)
Liver and Lung	2 (12.50%)
Omentum majus	2 (12.50%)

*One of CRC patient who had two lesions at both sides of colon was not included in subgroup analysis.

Diagnostic value of mSEPT9 ddPCR for CRC in peripheral blood

The average methylation index (MI) of SEPT9 in CRC patients was 2.21 ± 4.94%, with the highest MI as 26.68%. The diagnostic performance of mSEPT9 was assessed using ROC curve analysis, which revealed that mSEPT9 discriminated CRC from controls with an area under the curve (AUC) of 0.814 (95% CI, 0.737 to 0.892, p < 0.001) (Figure 2).The sensitivity and specificity of mSEPT9 detection in peripheral blood for diagnosis of all stages of CRC patients was 68.35% (54/79) and 95.00% (38/40), respectively.

Figure 2.

ROC curve analysis of mSEPT9 detection in CRC patients and controls.

For patients with colorectal adenoma and CRC of stage I to IV, positive rate of mSEPT9 detection in peripheral blood was shown in Table 4. The sensitivity for early-stage CRC (Stage I and II) and late-stage CRC (Stage III and IV) was 52.63% and 82.93%, respectively. The positive rate were similar between left-sided CRC and right-sided CRC (65.31% vs 72.41%，p ＞ 0.05).mSEPT9 detection in all healthy people with normal colonoscopy were negative.

Table 4.

Diagnostic value of mSEPT9 detection for CRC in peripheral blood.

Group of diseases	Positive rate
Healthy people (n = 20)	0
Colorectal adenoma (n = 20)	10.00%
Stage I CRC (n = 20)	35.00%
Stage II CRC (n = 18)	72.22%
Stage III CRC (n = 25)	80.00%
Stage IV CRC (n = 16)	87.50%
Location of lesions*
Left-sided CRC (n = 49)	65.31%
Right-sided CRC (n = 29)	72.41%

*One of CRC patient who had two lesions at both sides of colon was not included in subgroup analysis.

Performance of CEA and CA199 and combined detection of CEA, CA199 and mSEPT9 in peripheral blood for diagnosis of CRC

In CRC patients with available results of CEA and CA199, positive rate of CEA or CA199 was 38.67% and 22.67%，respectively. CEA or CA199 was elevated in 45.33% CRC patients. When mSEPT9 detection was combined, positive rate of any of CEA, CA199 or mSEPT9 was 82.67%. (Table 5)

Table 5.

Combined detection of CEA, CA199 and mSEPT9 in peripheral blood for diagnosis of CRC.

	Positive rate
Only CEA	38.67%
Only CA199	22.67%
CEA or CA199	45.33%
Any of CEA, CA199 or mSEPT9	82.67%

Discussion

This single-center study demonstrated that detection of mSEPT9 in peripheral blood could be a promising and non-invasive method for early diagnosis of CRC. Our findings indicated that the methylated SEPT9 assay by ddPCR method achieved a sensitivity of 68.35% and a specificity of 95.00% for all stages of CRC patients. When it was combined with CEA and CA199, the positive rate could be raised to 82.67%. These findings suggested that blood-based mSEPT9 detection could provide a new strategy for initial screening of CRC, especially for those unwilling or unable to undergo invasive procedures such as colonoscopy. In this study, the sensitivity for early-stage CRC was lower than expected, but it can be partially understood. As a single center study, the sample size was relatively limited, which may not be sufficient to provide an ideal result. Even so, the tendency of higher positive rate of mSEPT9 in more advanced stage of CRC was consistent with previous studies.¹⁵ It is particularly worth noting that the performance of mSEPT9 detection in diagnosis of CRC reported in previous studies vary greatly, which may be related to different sample size and detection methods used in different periods.

In general, the sensitivity for detecting CRC ranged from 48.2% to 95.6%, with the specificity ranged from 79.1% to 99.1%.^16–17 Church TR et al. prospectively assessed the accuracy of circulating mSEPT9 for detecting asymptomatic CRC in a screening population by using a commercially available assay (Epi proColon 1.0). In this earlier study, a standardized sensitivity of 48.2% and specificity of 91.5% was achieved, respectively.¹⁸ Potter NT et al. validated this real-time PCR–based qualitative assay in a prospective clinical trial, the sensitivity for all stages of CRC was 68% and adjusted specificity was 80.0%.¹⁹ While subsequent studies introduced quantitative PCR(qPCR)-based mSEPT9 commercial test kit with improved sensitivity and specificity. Recent years, Cai et al. developed a multilocus blood-based qPCR assay (ColonAiQ assay) and outperformed the current screening assays, with a sensitivity of 86% and specificity of 92%, respectively.⁹ The ColonAiQ assay integrated 6 markers that have undergone multiple rounds of screening, showing a detection rates of 65.1%, 82.7% and 81.5% of CRC stages I, II and III in a subsequent multicenter prospective cohort study.²⁰

Recent studies suggest that ddPCR has the potential for absolute quantification so that it is suitable for low-concentration samples. It can further improve the sensitivity of mSEPT9 assay for CRC screening and is superior to commercial qPCR kits. However, there are few studies on the detection of mSEPT9 in CRC by using ddPCR method. The general diagnostic performance observed in our study is consistent with previous studies. In particular, the high specificity of 95.00% in our study aligns with the notion that SEPT9 methylation is a cancer-specific event that rarely occurs in healthy people, which could minimize unnecessary colonoscopy. Furthermore, our data corroborate with previous findings that SEPT9 methylation is an early event in colorectal carcinogenesis, present even in colorectal adenomas, although with a lower detection rate.

As for the impact of location of tumor on the results, current research findings are not consistent. In this study, positive rate of mSEPT9 was similar between left-sided CRC and right-sided CRC. A recent study found that the SEPT9 methylated abundance was significantly higher in distal cancer, especially in sigmoid cancer.²¹ Given that colonoscopy performed in clinical practice include the examination of the entire colon and rectum, whether there are significant differences in the results of the left and right colon does not have a significant impact on clinical decision. According to previous studies, sensitivity and specificity of mSEPT9 in detecting CRC are better than those of fecal occult blood test, which relies on the intermittent bleeding of lesions. In addition, combined detection of mSEPT9 with fecal occult blood test and CEA and CA19-9 could improve the diagnostic sensitivity.^22–26 In this study, when mSEPT9 detection was combined with CEA and CA199, positive rate of detecting CRC could be raised to 82.67%. In addition to early diagnosis, mSEPT9 can also be used for prognosis evaluation and postoperative monitoring of CRC.^27–28 However, our study did not conduct subsequent follow-up tests on these patients because of resource constraints.

Our findings are substantial for clinical practice. Blood testing, as a method of liquid biopsy, is more convenient and non-invasive than colonoscopy. The process of collecting specimen for mSEPT9 detection could be integrated into routine check-up upon admission, which will not cause any additional discomfort to patients. Moreover, ddPCR is quantitative and more sensitive than traditional PCR so that the volume of plasma needed is much less than usual. By improving early detection rates and adherence of necessary colonoscopy, this assay has the potential to diagnose CRC at an earlier and more treatable stage, thereby reducing CRC-related mortality and improving quality of life. Furthermore, since only one locus is being tested, the cost is relatively lower, which facilitates more extensive screening as public health strategy.

There are some limitations that must be acknowledged in this study. First, it was a single-center study with limited participants. Secondly, mSEPT9 was only detected before surgery or endoscopic treatment, so we are unable to reveal its relationship with the prognosis or recurrence of disease in this exploratory study. In addition, due to the nature of research design, the diagnostic efficacy may be overestimated when compared to screening cohorts that include more asymptomatic individuals. Finally, the test did not detect other CRC methylation markers, which need complementary biomarkers such as CEA and CA19-9 to enhance its sensitivity further.

This was a pilot study and future research should undertake more work to address these issues. Multi-center studies with larger number of participants are needed to validate our findings. Moreover, large-scale prospective randomized trials comparing the mSEPT9 blood test with colonoscopy for first-line screening are essential to define its precise role in real world. In addition, cost-effectiveness analysis of a SEPT9-based screening strategy is also crucial for its widespread adoption by public healthcare systems.

Conclusion

In conclusion, this study provides evidence that blood-based mSEPT9 detection by ddPCR method is a sensitive and highly specific method for non-invasive diagnosis of CRC. Combination with CEA and CA19-9 could improve its performance. It may serve as an alternative option for individuals unwilling or unable to undergo invasive procedures.

Footnotes

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Suzhou Science and Technology Plan Project, (grant number SZM2022012).

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

ORCID iDs

Mengjie Lin

Caifeng Jiang

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