|Ahead of print
Quantitative detection of SRY-Box 21 (SOX21) gene promoter methylation as a stool-based noninvasive biomarker for early diagnosis of colorectal cancer by MethyLight method
Keivan Moradi1, Esmaeil Babaei2, Mohammad Ali Hosseinpour Feizi2, Reza Safaralizadeh2, Nayebali Rezvani3
1 Department of Clinical Biochemistry, Kermanshah University of Medical Sciences, Kermanshah; Department of Animal Biology, Faculty of Natural Science, University of Tabriz, Tabriz, Iran
2 Department of Animal Biology, Faculty of Natural Science, University of Tabriz, Tabriz, Iran
3 Department of Clinical Biochemistry, Kermanshah University of Medical Sciences, Kermanshah, Iran
|Date of Submission||09-Jan-2019|
|Date of Decision||12-Jul-2019|
|Date of Acceptance||22-Jul-2019|
Department of Clinical Biochemistry, Kermanshah University of Medical Sciences, Kermanshah
Source of Support: None, Conflict of Interest: None
Background: In recent years, the study of potential epigenetic biomarkers in feces has been an attractive research approach for the noninvasive diagnosis of colorectal cancer (CRC). The aim of this study was to evaluate the stool-based DNA methylation potential of SRY-Box 21 (SOX21) gene promoter as an appropriate candidate biomarker for differentiating CRC patients and healthy individuals for the first time.
Methods: The MethyLight method was performed to analyze the methylation status of SOX21 gene promoter in fecal samples from 40 patients with CRC and 40 healthy controls. In addition, the diagnostic efficiency of measuring the hypermethylated SOX21 gene in the feces to the fecal occult blood test (FOBT) was compared.
Results: The percentage of methylated reference (PMR) values in the stool of CRC patients (median 1.44) was higher than those of healthy individuals (median 0.00) (P < 0.001). A sensitivity of 72.5% and specificity of 100% were obtained for SOX21 gene promoter methylation status and 29 of the patients were considered as positive in methylation status. There was no significant association between PMR values and demographic/clinicopathological features (P > 0.05).
Conclusion: The results of the present study demonstrated that the stool-based assay of SOX21 gene promoter methylation has a relatively high sensitivity and specificity and it may serve as a noninvasive biomarker for early detection of CRC. However, more studies with a wide range of samples are required to further confirm the role of hypermethylation of SOX21 in the early CRC diagnosis.
Keywords: Biomarker, colorectal cancer, early detection, methylation, stool
Key Message: Noninvasive diagnosis of colorectal cancer (CRC) significantly increases the patients' survival rate. Stool-based assay of SRY-Box 21 SOX21 gene promoter methylation has the potential to serve as a noninvasive biomarker for early detection of CRC.
|How to cite this URL:|
Moradi K, Babaei E, Hosseinpour Feizi MA, Safaralizadeh R, Rezvani N. Quantitative detection of SRY-Box 21 (SOX21) gene promoter methylation as a stool-based noninvasive biomarker for early diagnosis of colorectal cancer by MethyLight method. Indian J Cancer [Epub ahead of print] [cited 2020 Oct 30]. Available from: https://www.indianjcancer.com/preprintarticle.asp?id=297022
| » Introduction|| |
Colorectal cancer (CRC) is the third most common cancer and the fourth leading cause of cancer-associated mortality throughout the world. This type of cancer affects both men and women fairly equally and over half a million deaths occur each year from the disease., The stage in which the disease is diagnosed greatly affects the survival of patients. The 5-year survival rate increases to more than 90% diagnosed in the early stages of the disease. Therefore, the use of appropriate screening methods can detect CRC at the early stages and reduce mortality.
Colonoscopy and fecal occult blood test (FOBT) are currently the most common methods for the screening of CRC. Despite the excellent diagnostic accuracy of colonoscopy (sensitivity of 94.7% and specificity of 99.8%) and its ability to remove polyps and precancerous lesions, some limitations such as high cost and the invasive nature of the method have diminished its popularity., On the other hand, despite the benefits such as low cost and noninvasive nature of FOBT, the high false positive and false negative rates have reduced the credibility of the test.
Studies performed in recent years have shown that epigenetic changes in certain genes have the potential for use as biomarkers for early diagnosis of cancer. The occurrence of methylation in the CpG islands is an epigenetic change that has been reported in various cancers. In the human genome, about two-thirds of all promoters contain CpG islands. The addition of a methyl group to the cytosine bases located in CpG islands in the promoter of tumor suppressor genes results in silencing of these genes.
SOX21 (SRY-Box 21) is a candidate tumor-suppressor gene found in a number of cancer types. This gene is located on chromosome 13q31-q32 and encodes a transcription factor. By studying a panel of genes, Mitchell et al., showed that the promoter of this gene was hypermethylated in plasma (compared to normal controls) and tumor tissues (compared to adjacent normal tissues) of CRC patients. Therefore, these authors suggested SOX21 gene promoter as a good candidate biomarker for stool-based assay. In addition, according to a recent study performed by us,SOX21 gene promoter methylation showed a sensitivity of 80.5% and a specificity of 97.6%. Our results were comparable to the sensitivity and specificity of most robust DNA methylation-based biomarkers reviewed by Lam et al.
In recent years, the study of the sensitivity and specificity of potential epigenetic biomarkers in feces has been an attractive research approach for noninvasive diagnosis of CRC.,, Therefore, we conducted the present study for the first time to evaluate the stool-based DNA methylation potential of SOX21 gene promoter as an appropriate candidate biomarker for differentiating CRC patients and healthy individuals. In addition, the FOBT was performed for all participants in the study, and the sensitivity and specificity of the test were evaluated.
| » Materials and Methods|| |
Subjects and specimens
This study was approved by the Ethics Committee of Kermanshah University of Medical Sciences, Kermanshah, Iran (project code: 95285). All individuals participated voluntarily in the study and a written informed consent was obtained from all of them. A total of 80 participants, including 40 CRC patients and 40 healthy controls, participated in the study. The sample size calculation was based on the papers published previously in the literature. All patients had undergone surgery at Bisotun Hospital (Kermanshah, Iran) in a 3-year period (2016–2018). Cancers were staged according to the cancer staging manual (7th edition) from the American Joint Committee on Cancer (AJCC). The criteria for choosing healthy controls were based on the negative results of colonoscopy and FOBT, no history of malignancy or inflammatory bowel disease, no history of chemotherapy or radiotherapy, and satisfaction to participate in the study. On the other hand, the criteria for choosing CRC patients were based on positive results of colonoscopy and pathology tests, no history of chemotherapy or radiotherapy, and satisfaction to participate in the study. Stool specimens of all the participants were collected prior to any surgical or chemotherapy intervention. All of the different stages of the study including stool sampling, FOBT, and the measurement of methylation were performed at the Reference Laboratory, Kermanshah, Iran. Each of the stool specimens was immediately divided into two parts: One part was kept at a temperature of −80°C until the DNA extraction step was carried out, and the FOBT was performed on the other part.
DNA extraction and bisulfite treatment
Three to four days after collection of specimens, the genomic DNA was extracted from stool samples (20 mg) with the QIAamp DNA Stool Mini Kit (Qiagen, Germany; cat no. 51104) according to the manufacturer's instructions. The extracted DNA was quantitatively and qualitatively evaluated using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Inc., Wilmington, DE, USA). Genomic treatment by sodium bisulfite results in the conversion of unmethylated cytosine residues to uracil, while leaving the methylated cytosines at CpG sites unchanged. After the modification of 500ng of genomic DNA by using of the EpiTectH Bisulfite Kit (Qiagen, Germany; cat no. 59826) according to the manufacturer's protocol, the bisulfite-treated DNA was stored at −20°C until being processed.
In this study, the MethyLight method was used to evaluate the methylation level of the SOX21 gene promoter. To detect methylated DNA sequences from bisulfite-treated templates, a pair of primers and a TaqMan probe specified to anneal the fully DNA methylated at SOX21 gene promoter were designed using Beacon Designer™ (version 8.13; www.premierbiosoft.com/molecular_beacons; Premier Biosoft International, Palo Alto, CA, USA); forward primer: 5'-CGTCGGGGCGCGACGAGTT-3', reverse primer: 5'-ACTCTCGCCCTACCGCGACTAC-3', and probe: 6FAM 5'-CCGCCTCCCGAACCCTCGCCCT-3'TAMRA. The MethyLight reactions were performed on a 7500 real-time polymerase chain reaction (real-time PCR) system (Applied Biosystems, Thermo Fisher Scientific, Inc.) using the EpiTect® MethyLight PCR + ROX Vial kit (Qiagen GmbH; cat. no. 59496) according to the manufacturer's protocol. The final volume of each reaction was 20 μl containing 2 μl of 10X primer-probe mix containing 200 nM probe and 400 nM of each forward and reverse primers, 10 μl of 2X EpiTect MethyLight master mix (without ROX), 0.4 μl of 50X ROX dye solution, 2 μl (20 ng) of bisulfite-treated DNA template, and 5.6 μl of RNase-free water. The PCR conditions were: 95°C for 3 minutes, followed by 50 cycles at 95°C for 5 seconds, 64°C for 10 seconds, and 73°C for 20 seconds. The ALU-C4 element was used as a reference control using the primer and probe sequences as previously described.
To establish the standard curve, a fully methylated control (EpiTect PCR Control DNA; Qiagen GmbH; cat. no. 59655) was used to calculate the percentage of methylated reference (PMR) of the samples. PMR is the degree of methylation of each sample relative to the fully methylated control and was calculated using the following formula: [(SOX21/ALUC4) sample/(SOX21/ALUC4) positive control] ×100.
Fecal occult blood test (FOBT)
Guaiac test is the most common type of FOBT and looks for hidden (occult) blood in a stool sample. The guaiac test was used in the present study according to the protocol previously described.
Statistical data analysis was carried out using SPSS software (version 25.0; SPSS, Inc., Chicago, IL, USA). The P values ≤0.05 were considered significant for all described tests. 2-sided Mann–Whitney U-test was used to compare the median level of methylation in CRC and normal fecal stool samples. To measure the association between classified clinicopathological characteristics with the median level of methylation, the Fisher's exact test and 2-sided Mann–Whitney U-test were used. To assess the sensitivity and specificity, a receiver operating characteristics (ROC) curve was constructed. To differentiate patients based on their methylation status, the point on the ROC curve at which the sum of sensitivity and specificity was maximum was considered as the optimal cut-off PMR value. The patients were classified as positive or negative according to having PMR more than or less than the cut-off PMR value, respectively.
| » Results|| |
To investigate the potential of SOX21 gene promoter methylation as a suitable biomarker for early detection of CRC, we extracted the genomic DNA in the stool of 40 patients with histologically diagnosed CRC and 40 healthy individuals. The median age of CRC patients and healthy individuals was 55 years (range: 31–80) and 51.5 years (range: 34-84), respectively. The ratio of male/female in the patient and control groups was 0.90 (19/21) and 0.74 (17/23), respectively. The standard curves and the results of real-time PCR for amplification of SOX21-methylated and ALU-C4 (internal control) from some of the patients and controls are shown in [Figure 1]. Statistical results showed that there was no significant difference between age and gender in the patient and control groups. The demographic/clinicopathological characteristics of the individuals participated in the study are shown in [Table 1].
|Figure 1: Standard curves for SOX21 gene promoter methylation and ALU-C4 element (a and b, respectively), as well as the results of real-time polymerase chain reaction (PCR) for the amplification of these elements (c and d, respectively) obtained from some of the patient and control samples|
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|Table 1: Demographic/clinicopathological characteristics of the individuals participated in the present study|
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The methylation level of SOX21 gene promoter was measured in both groups of patients with CRC and healthy individuals using the MethyLight technique. The Mann–Whitney U-test showed that the PMR values in the stool of CRC patients (median: 1.44) were higher than those of healthy individuals (median: 0.00) (P < 0.001) [Table 2].
|Table 2: PMR results for SOX21 gene promoter methylation in the stool of patients with CRC and healthy individuals|
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The relationship between the demographic/clinicopathological features and the methylation level of SOX21 gene promoter in the stool of the patient group is summarized in [Table 3]. According to the obtained results, there was no significant association between PMR values and demographic/clinicopathological features (P > 0.05).
|Table 3: Association of SOX21 gene promoter methylation with characteristics in patients with CRC|
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By using the optimal cut-off PMR value (0.4), stool samples exhibited a sensitivity of 72.5% and specificity of 100% for SOX21 gene promoter methylation status, and 29 (72.5%) and 11 (27.5%) of the patients were considered as positive and negative in methylation status, respectively. An area under the curve (AUC) of 0.875 (95% confidence interval (CI) 0.792–0.959) was calculated by the SPSS software [Figure 2].
|Figure 2: Receiver operating characteristics (ROC) curve for SOX21 gene promoter methylation|
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FOBT was examined in CRC patients and control group. The obtained sensitivity and specificity for this fecal test were 47.5% and 100%, respectively [Table 4].
|Table 4: The positive rate of hypermethylated SOX21 and FOBT in the stool samples of CRC patients and control group|
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| » Discussion|| |
It has been shown that the tumor cells of the intestinal layer can be isolated and released into the feces and it has been proposed to use molecular tests to investigate genetic and epigenetic changes in the fecal DNA as noninvasive screening methods for early detection of CRC.
In recent years, many studies have been conducted on the identification and description of methylation-based biomarkers in CRC patients in diagnostic, prognostic, and therapy prediction areas. Cologuard (a multitarget stool DNA test including two DNA methylation biomarkers and seven DNA mutation biomarkers) and Epi proColon (a blood-based test for methylated SEPT9 DNA) are two noninvasive DNA tests for CRC screening that have recently been approved by the Food and Drug Administration.
SOX21 is a candidate tumor-suppressor gene found in a number of cancer types. SOX21 was seen to be downregulated in high-grade glioma compared to low-grade glioma. It has been shown that the expression of SOX21 and SOX2 proteins overlap overlaps in glioma tissues and cell lines. Unlike the oncogenic activity of SOX2, SOX21 overexpression decreases proliferation and induces apoptosis., In addition to glioma, Kurtsdotter et al. have found that the SOX21 mRNA levels are significantly downregulated in human gastric cancer compared to normal tissue. These authors also reported that by overexpressing SOX21 in human gastric cancer cell lines, proliferation decreases and apoptosis is induced.
According to a study conducted by Kandimalla et al.,SOX21 gene promoter is hypermethylated in bladder tumors but not in control urine-derived DNA. SOX21 gene promoter methylation was first reported by Mitchell et al. in 2014 as a candidate biomarker for stool-based assays in CRC patients. These authors showed that 82% of colon tissue samples (67 samples) and 0% of matched normal tissues (6 samples) were hypermethylated in CRC patients. In addition, in their second study, Mitchell et al. reported a sensitivity of 85% in the plasma samples of CRC patients. Moreover, our previous study on the status of SOX21 gene promoter methylation in CRC tissues and their normal adjacent mucosa showed a sensitivity of 80.5% and a specificity of 97.6%.
In this study, we investigated the state of methylation of SOX21 gene promoter in the stool samples of 40 patients with CRC and 40 healthy individuals for the first time. Our results showed an AUC of 0.875, a sensitivity of 72.5%, and a specificity of 100% based on the optimal PMR value mentioned in the methods section. PMR values in the stool of CRC patients were significantly higher than those of healthy individuals (P < 0.001). The results of this study, along with our previous study and the two studies conducted by Mitchell et al.,, are shown in [Table 5]. The lower sensitivity observed here compared to the sensitivities reported in other studies can be attributed to the difference in the type of examined samples (fecal samples compared to tissue and plasma samples). However, the results obtained in this study confirm the high potential of SOX21 gene promoter methylation as a candidate biomarker in the stool samples of CRC patients.
|Table 5: Comparison of the results of the previous studies on SOX21 gene promoter methylation status with the results obtained by us|
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By studying the literature, it was revealed that there is no report of the relationship between the methylation status of SOX21 gene promoter and demographic/clinicopathological features in stool samples of CRC patients. Therefore, we categorized patients' demographic/clinicopathological features into subgroups and examined their association with the median PMR values obtained from stool samples in each subgroup. No statistically significant relationship was identified between SOX21 gene promoter methylation levels and and the age and gender of CRC patients, tumor type, histological grade of differentiation, TNM stage, tumor location, or tumor size.
Currently, FOBT is the most commonly used noninvasive screening test for CRC.,, However, according to a review article conducted by Allameh et al., the mean sensitivity of FOBT is 45.7%. Therefore, the obtained sensitivity for FOBT in the present study (47.5%) is compatible with the Allameh et al. study. In addition, the higher sensitivity of SOX21 gene promoter methylation in CRC stool samples compared to FOBT sensitivity indicates its high potential as a candidate biomarker. On the other hand, the sensitivity and specificity of colonoscopy is 94.7% and 99.8%, respectively. Therefore, although the sensitivity of stool-based SOX21 gene promoter methylation is less than that of colonoscopy (72.5% compared to 94.7%), their specificity is similar (100% compared to 99.8%). In addition, since all of our patients were colonoscopy-positive and all of our controls were colonoscopy-negative, both of sensitivity and specificity of colonoscopy in this study were 100%.
The clinical implications of the results of this study can be summarized as follows. First, unlike the colonoscopy as an invasive CRC detecting method,, the analysis of SOX21 gene promoter methylation in the stool samples of CRC patients is a noninvasive method. Second, it has been shown that colonoscopy is less effective in detecting proximal neoplasms than distal neoplasms,,, while the efficiency of stool-based SOX21 gene promoter in detecting of these two neoplastic groups was not significantly different (P > 0.05). Third, the obtained sensitivity of stool-based SOX21 gene promoter (72.5%) for early detection of CRC is higher than the sensitivity of FOBT (47.5%) that is routinely performed in clinical laboratories.
| » Conclusion|| |
In summary, the results of the present study demonstrated that the stool-based assay of SOX21 gene promoter methylation has relatively high sensitivity and specificity and it may serve as a noninvasive biomarker for early detection of CRC. However, more studies with a wide range of samples are required to further confirm the role of hypermethylation of SOX21 in the early CRC diagnosis.
Declaration of patient consent
The authors certify that they have obtained all appropriate patient consent forms. In the form, the patients/healthy individuals have given their consent for their images and other clinical information to be reported in the journal. The patients/healthy individuals understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
The authors would like to thank the staff of the Medical Genetics Laboratory, Reference Laboratory, Kermanshah University of Medical Sciences, for their assistance during the present study.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]