Evaluating the cross-reactivity of a stool methylated syndecan-2 (meSDC2) test in colorectal cancer detection

Introduction

Colorectal cancer (CRC) ranks as the third most commonly diagnosed cancer and is the second leading cause of cancer-related death worldwide (1,2). The 5-year survival rate for CRC ranges from 90% for patients diagnosed at early stages to 14% for those diagnosed with advanced stages (3). Therefore, early detection of CRC is crucial for improving patient survival. Currently, sequential screening programs for early detection of CRC are recommended, including a fecal occult blood test followed by a full colonoscopy. A non-invasive fecal immunochemical test has been used for the primary screening of CRC. However, despite its relatively favorable performance characteristics, fecal immunochemical test (FIT) exhibits suboptimal sensitivity for detecting early-stage CRC and advanced adenomas, and even modest false-positive rates can have significant clinical implications in large-scale screening (4). Although colonoscopy is considered the gold standard for the early detection of CRC, its compliance rate is low due to patient discomfort and the required bowel preparation (5). Therefore, developing simple, cost-effective and highly accurate non-invasive molecular biomarker tests in bodily fluids are necessary for the primary screening of CRC in patients who are reluctant to undergo colonoscopy.

DNA methylation, a crucial epigenetic phenomenon, plays a fundamental role in various biological processes, including development, cell differentiation, ageing, tumorigenesis, and other diseases (6,7). Aberrant DNA methylation is involved in tumour development and is considered one of the earliest and most frequent genomic alterations occurring during carcinogenesis (8,9). Furthermore, the fact that aberrant DNA methylation occurs extensively throughout the genome allows for improved detection sensitivity and specificity, as multiple methylation targets can be assessed simultaneously in a single reaction (10). Therefore, DNA methylation alterations in cancer can provide a unique opportunity for screening, early detection, prediction, and prognosis of cancer through methylation analysis using body fluids (6,10,11). To date, considerable efforts have been dedicated to developing DNA methylation biomarkers for the early detection of cancer.

Several previous studies have revealed that methylated syndecan-2 (meSDC2) has high potential as a stool DNA-based diagnostic biomarker for early CRC detection (12-15). In addition to CRC, meSDC2 has been identified in gastric tumours (16,17), head and neck squamous cell cancer (18), and glioma tissues (19). Maintaining a high specificity is essential for reducing false positives and unnecessary follow-up procedures during early cancer detection. Even a low false-positive rate can significantly impact many patients in clinical practice, leading to avoidable and costly diagnostic procedures (20). Since tumours in organs connected to the digestive tract may release tumour-derived methylated DNA into stool samples, assessing the potential cross-reactivity of the meSDC2 test with other cancers is imperative.

In this study, we aimed to investigate the specificity of the stool DNA-based meSDC2 test for CRC. We tested patients with various types of cancers who showed no symptoms of colorectal neoplasia. The study comprehensively explored the methylation patterns of the meSDC2 biomarker in multi-cancer DNA samples obtained from stool samples of patients with 13 distinct types of potentially interfering cancer, including CRC. Our goal was to evaluate the ability of the meSDC2 test to specifically detect CRC in stool samples while maintaining a low false-positive rate. We present this article in accordance with the STARD reporting checklist (available at https://jgo.amegroups.com/article/view/10.21037/jgo-2025-270/rc).

Methods

Patient enrolment and sample collection

This study was conducted at the Asan Medical Center, Seoul, South Korea. We enrolled patients with hepato-gastroenterological cancers (gastric cancer, liver cancer, oesophageal cancer, pancreatic cancer, biliary tract cancer) and other malignancies such as breast, cervical, lung, bladder, prostate, kidney, and renal pelvis cancers. All cancer cases were confirmed histopathologically by board-certified pathologists at Asan Medical Center. The clinicopathological and demographic information of the included patients was prospectively recorded. To analyse fecal meSDC2, at least 2 g of stool samples were collected from four to five different locations using a stool collection kit (Genomictree, Inc., Daejeon, South Korea). All stool samples were collected before therapeutic intervention and sent to a central laboratory for methylation analysis. Patients with CRC were included as a positive control. Apart from patients with CRC, individuals with other types of cancer did not undergo colonoscopy.

The detailed criteria for enrolment in this study were as follows: (I) patients who voluntarily signed an informed consent form; (II) patients aged 19–79 years; (III) patients with no history of cancer; (IV) individuals who had not received treatment (surgery, chemotherapy, hormone therapy, and radiation therapy) after being diagnosed with cancer; (V) patients who agreed to provide stool specimens; and (VI) patients who agreed to provide demographic information, imaging, and histology results. Patients were excluded from the study if they met any of the following criteria: (I) individuals who did not agree to voluntarily sign an informed consent; (II) patients with a history of cancer; (III) patients diagnosed with two types of cancer simultaneously; (IV) individuals who had received any treatment (surgery, chemotherapy, hormone therapy, and radiation therapy) after being diagnosed with cancer; (V) patients who did not agree to provide stool and urine specimens; and (VI) patients with severe comorbidities, poor performance status, or any acute clinical condition that, in the opinion of the investigator, would place the patient at excessive risk from study procedures. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. This study was approved by the Institutional Review Board at the Asan Medical Center (IRB No. 2022-0704; approval date: 2022-05-26), and written informed consent was obtained from all individuals who agreed to participate.

Sample handling process and assessment of meSDC2 in stool

Stool DNA was isolated using either the solid-phase magnetic bead-based GT NUCLEIC ACID PREP Kit II (Genomictree, Inc.) or the Chemagic 360-D instrument with the Chemagic DNA Stool 3k Kit H24 (PerkinElmer, Waltham, MA, USA) according to the manufacturer’s instructions. The Qubit dsDNA BR assay kit (Thermo Fisher Scientific, Waltham, MA, USA) was used to determine the DNA concentration of the stool sample. Each 2.0 µg of stool-derived genomic DNA was chemically modified with sodium bisulphite using the EZ DNA Methylation-Gold Kit (Zymo Research, Irvine, CA, USA) according to the manufacturer’s instructions. The bisulfite-converted DNA was purified and eluted with 12 µL of elution buffer using a Zymo-Spin IC column (Zymo Research) and immediately used for the meSDC2 test.

To measure meSDC2 in stool DNA, the two-step linear target enrichment (LTE)/quantitative methylation-specific polymerase chain reaction (qMSP) was performed in two polymerase chain reaction (PCR) reactions for each sample as previously described (12). The region lacking CpG dinucleotides of the COL2A1 gene was also used as a control. The LTE/qMSP meSDC2 test was performed on the AB 7500 Fast Dx Real-Time PCR (Thermo Fisher Scientific). A total of 20 µL of reaction mixture containing 2.0 µg of bisulfite-converted stool DNA, 50 nmol/L each of meSDC2-specific antisense (5'-ACGACTCAAACTCGAAAACTCG-3') and COL2A1 gene-specific antisense primers (5'-CTAICCCAAAAAAACCCAATCCTA-3') attached to 5' universal tag sequence (5'-AAAGATTCGGCGACCACCGA-3'), and 4 µL of 5× AptaTaq PCR master mix (Roche Diagnostics, Basel, Switzerland) was prepared. Thermal cycling conditions were as follows: 95 ℃ for 5 min followed by 35 cycles of 95 ℃ for 15 s and 60 ℃ for 60 s.

After LTE, the reaction mixture volume was scaled up to 40 µL, containing 8 µL of 5× AptaTaq PCR master mix, 250 nmol/L of meSDC2-specific sense primer (5'-GTAGAAATTAATAAGTGAGAGGGC-3') 125 nmol/L of SDC2 probe (5'-FAM-TTCGGGGCGTAGTTGCGGGCGG-3'), 125 nmol/L of COL2A1 sense primer (5′-GTAATGTTAGGAGTATTTTGTGGITA-3'), 62.5 nmol/L of COL2A1 probe (5'-Cy5-AGAAGAAGGGAGGGGTGTTAGGAGAGG-3'), and 250 nmol/L of universal sequence primer. Thermal cycling conditions were as follows: 95 ℃ for 5 min followed by 40 cycles of 95 ℃ for 15 s and 60 ℃ for 60 s. Heating and cooling rates were 20 ℃ per second and 15 ℃ per second, respectively.

For PCR analysis, meSDC2 and COL2A1 control reactions were run in a single tube. For each run, bisulfite-converted methylated (HCT116 cells, ATCC, Manassas, VA, USA) and unmethylated genomic DNA (Genomictree, Inc.) were used as methylation controls. Non-template and non-template bisulphite-converted controls were also included.

Cycle threshold (C_T) values were calculated using the real-time PCR 7500 software version 2.3 (Thermo Fisher Scientific). meSDC2 was considered detected if the C_T value was <40 cycles. If the C_T value was not measurable, methylation was not detected. Samples were deemed positive if methylation was detected in both reactions and negative if not detected in either reaction. Test results were labelled inconclusive if methylation was detected in only one of the two reactions, prompting a retest. If methylation was detected in at least one of the retest reactions, the sample was considered positive; if the retest was negative in both reactions, the sample was considered negative. Acceptable test results required the C_T value of COL2A1 to be less than 33, indicating the limit of detection for the COL2A1 gene (data not shown) in both reactions. The process of evaluating meSDC2 was schematised in Figure 1.

Figure 1 Schema for assessing meSDC2 in stool-based DNA. +, positive; −, negative. C_T, cycle threshold; meSDC2, methylated syndecan-2.

Statistical analysis

All statistical analyses were performed using MedCalc V20.015 (MedCalc Software Ltd., Ostend, Belgium). To calculate methylation-positive rates, test results were dichotomised as ‘1’ for methylation-positive and ‘0’ for methylation-negative. Demographic and other clinical characteristics are described as frequencies and percentages. We investigated the cancer incidence rates using Korean cancer statistics to estimate the predicted positivity rates for each cancer type (21). The result was considered statistically significant if the P value was <0.05.

Results

Patient enrolment

We enrolled 190 patients with various types of cancer from May 2022 to April 2023. Four patients were excluded due to inappropriate sample collection timing or cases where samples were lost after collection, and two patients were excluded because the final pathological results were identified as benign lesions. A total of 184 patients were finally included in the study. The number of patients and demographic information for each cancer group are presented in Table 1.

Positivity of meSDC2 stool test by cancer type

Out of the 184 enrolled patients, valid stool test results were obtained from 168. Among the patients with CRC serving as a positive control, 17 out of 19 were positive, yielding a positivity rate of 89.5%. The positivity rate of the meSDC2 stool test was 15.8% (3/19) for gastric cancer and 22.2% (4/18) for oesophageal cancer. Additionally, biliary tract and pancreatic cancers showed positivity rates of 44.4% (4/9) and 28.6% (2/7), respectively. Conversely, among non-hepato-gastroenterological cancers, the highest rate was observed in prostate cancer patients at 31.6% (6/19), and the lowest in breast cancer patients at 12.5% (1/8). The positivity rate for each cancer type is shown in Figure 2. The positivity rates of the stool meSDC2 test according to cancer type and tumor stage are presented in Table 2.

Figure 2 Positivity rate of meSDC2 in stool-based DNA for each type of cancer (in %). meSDC2, methylated syndecan-2.

Expected positive rate of stool meSDC2 test per 10,000 persons by cancer type

Based on the cancer registry statistics in South Korea, the incidence rate per 10,000 persons for each cancer type was retrieved, and the expected positive rate of the stool meSDC2 test per 10,000 persons was calculated. Excluding CRC, which served as a positive control, the highest expected positive rate was observed in lung cancer at 1.13 individuals per 10,000 persons, followed by prostate cancer at 1.03 individuals per 10,000 persons. For all other cancer types, excluding lung and prostate cancer, the expected positive rates were <1 individual per 10,000 persons. The results of the expected positive rates by cancer type are presented in Figure 3.

Figure 3 Expected number of positive cases per 10,000 individuals for meSDC2 in stool-based DNA by each type of cancer, n per 10,000 persons. meSDC2, methylated syndecan-2.

Discussion

We aimed to evaluate the cross-reactivity of the stool meSDC2 test in cancers other than CRC. As expected, the meSDC2 test showed a high positivity rate in CRC, which served as a positive control. On the other hand, the stool meSDC2 test showed a low positivity rate in other types of cancer, excluding biliary tract cancer. Biliary tract cancer among hepato-gastrointestinal cancers and prostate cancer among non-hepato-gastrointestinal cancers showed positivity rates greater than 30%. Considering the incidence rate per 10,000 people, the predicted positive rate is deemed to be negligible. Therefore, the results of this study indicate that the stool meSDC2 test is highly specific for CRC.

SDC2 is a protein component of the cell membrane, characteristically expressed in mesenchymal cells but not in normal intestinal epithelial cells (22). Previous clinical studies with the stool meSDC2 test demonstrated high sensitivity and specificity in diagnosing CRC (12-15). Despite the remarkable performance of this new stool-based test for CRC diagnosis, little attention has been paid to its cross-reactivity with other cancers in previous research. Only one study evaluated the positivity rate of this test in patients with gastric and liver cancers (12). Our study stands out for assessing the positivity rate of this test in 12 different non-CRC cancer types. In previous studies using neoplastic tissues, meSDC2 was found in gastric cancer (16), head and neck cancer (18), and glioma tissues (19), but to our knowledge, no research findings have addressed other types of cancer. Hence, evaluating its cross-reactivity with other types of cancer is necessary to establish stool meSDC2 as a biomarker for CRC diagnosis, which this study aimed to confirm. Our study showed a 15.8% positivity rate among patients with gastric cancer. In South Korea, the incidence of gastric cancer is relatively high, with age-standardized rates around 30–40 per 100,000 annually (23). However, due to a nationwide screening program that provides biennial upper endoscopy to individuals aged 40 years and older, more than 60% of newly diagnosed gastric cancers are detected at stage I (21,24). Consistent with this, 14 of 18 included gastric cancer patients in our study had stage I disease. Given this low prevalence and predominance of early-stage cancer, the expected positive rate of stool testing due to gastric cancer is estimated to be well below 1 per 10,000 individuals, and thus is not anticipated to significantly impact the application of the stool meSDC2 test in CRC screening.

Another stool-based CRC screening test, the multi-target DNA test (Cologuard, Exact Sciences, Madison, WI, USA), has been evaluated for cross-reactivity in non-CRC cancers. In that study, the positive rates were 41.6%, 50%, and 25% for pancreatic, liver, and gastric cancers, respectively (25). These figures are notably higher than those observed in our study. Additionally, similar to our analysis method, when considering the incidence rate of each cancer type per 10,000 individuals, the expected positivity rate was not significantly different from our results (25). This suggests that the specificity for CRC of this stool-based meSDC2 test is likely to be minimally affected by non-CRC cancers. Nevertheless, in our study, the positivity rate of the stool meSDC2 test in patients with biliary cancer was 44.4%. Since the study participants were not required to undergo colonoscopy, it was not possible to confirm the presence or absence of synchronous colorectal neoplasia, and only nine patients with biliary cancer were included in this study, so the association between this stool-based test and biliary cancer could not be defined. In previous studies, the role of SDC2 in cholangiocarcinoma has not been well understood, with only a few studies examining its level of expression in tissues to date. Further research is required to investigate the relationship between biliary tract cancer and stool-based meSDC2 tests in the future. Findings from such studies will be critical in guiding the clinical interpretation of positive meSDC2 results in individuals with no colorectal neoplasia on colonoscopy. Additionally, a low but consistent positivity rate was observed in patients with non-digestive malignancies, which gives rise to two potential interpretations. First, as colonoscopy was not performed in these individuals, the presence of undetected colorectal neoplasia cannot be entirely excluded. Alternatively, this finding may suggest a limitation in the tumor specificity of meSDC2 as a CRC biomarker, thereby warranting further validation in large-scale, prospective studies.

This study also has several limitations. First, the number of patients for each cancer type was relatively small. Due to the limited number of patients, assessing the positivity rate according to the stage or clinical information of each cancer type was challenging. The second limitation is that, with the exception of patients with CRC, none of the patients with other cancer types underwent colonoscopies. Consequently, the presence of synchronous colonic neoplastic lesions, such as CRC or advanced adenomas, which could potentially influence the results of the stool-based meSDC2 test, cannot be excluded. In follow-up studies, comparing the results of the stool meSDC2 test with colonoscopy findings in patients with biliary tract and pancreatic cancer, both of which showed positivity rates exceeding 30% in this study, would be of great value to assess the validity of potential cross-reactivity. Third, although CRC screening is typically recommended for individuals aged 50 years and older, the mean age of participants in this study was 64.9 years, suggesting that the study population was broadly representative of the screening-eligible age group. Nevertheless, since screening programs are designed for asymptomatic individuals, the absence of symptom assessment at the time of stool collection constitutes a limitation of this study. Fourth, in our study, five out of nine patients with biliary tract cancer were diagnosed with stage IV disease. Given that advanced-stage tumors are known to shed greater amounts of tumor DNA, this likely contributed to the relatively high positivity rate observed in this group. Therefore, the overrepresentation of late-stage biliary tract cancers in our cohort may have influenced the test performance and should be considered a limitation when interpreting the cross-reactivity findings. Lastly, due to the nature of the study population, in which most patients were being treated for other primary malignancies, follow-up colonoscopy was not consistently performed at Asan Medical Center and could not be reliably tracked for those referred to external centers. This limits our ability to assess post-test confirmation of colorectal neoplasia following a positive meSDC2 result.

Conclusions

This study has demonstrated that the stool-based meSDC2 test exhibits high specificity for CRC. Our results support the significant value of using this test for CRC screening. Further prospective studies with large-scale populations are required to evaluate the utility of the stool-based meSDC2 test as a screening tool for CRC.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the STARD reporting checklist. Available at https://jgo.amegroups.com/article/view/10.21037/jgo-2025-270/rc

Data Sharing Statement: Available at https://jgo.amegroups.com/article/view/10.21037/jgo-2025-270/dss

Peer Review File: Available at https://jgo.amegroups.com/article/view/10.21037/jgo-2025-270/prf

Funding: This study was supported by a research grant from Genomictree, Inc.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jgo.amegroups.com/article/view/10.21037/jgo-2025-270/coif). T.J.O. and S.A. are employees of Genomictree, Inc. (Daejeon, Republic of Korea). The other authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. This study was approved by the Institutional Review Board at the Asan Medical Center (IRB No. 2022-0704; approval date: 2022-05-26), and written informed consent was obtained from all individuals who agreed to participate.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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