Evaluation of FOXF1 promoter DNA methylation, as a promising epigenetic biomarker for colorectal cancer in stool samples

Evaluation of FOXF1 promoter DNA methylation, as a promising epigenetic biomarker for colorectal cancer in stool samples | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Evaluation of FOXF1 promoter DNA methylation, as a promising epigenetic biomarker for colorectal cancer in stool samples Susan Tahmasebi, Bayan Mehrjoei, Asad Vaisi-Raygani, Nayebali Rezvani This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5551296/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Colorectal cancer (CRC) pathogenesis is influenced by genes promoter region methylation, a key epigenetic mechanism. We sought to determine whether FOXF1 promoter methylation in stool samples could serve as a CRC biomarker. Materials and methods: We analyzed FOXF1 gene promoter methylation in stool samples from 50 CRC patients and 50 healthy controls. The percentage of methylation reference (PMR) value was determined for each sample. Results: PMR levels of the FOXF1 gene were higher in CRC patients than in controls (P<0.001). Median PMR values were 0.06 (95% CI 0.01-13.1) in stool samples from CRC patients and 0.01 (95% CI 0.0-0.02) in controls. Receiver Operating Characteristics (ROC) Curve analysis displayed a sensitivity of 59% and specificity 96% for FOXF1 gene methylation in stool samples. Conclusion: The elevated FOXF1 methylation levels in CRC stool samples, along with the test's sensitivity and specificity, suggest that this gene may be utilized as a non-invasive biomarker for colorectal cancer detection, independent of sex, age, or disease stage. Colorectal cancer Epigenetic Biomarkers DNA methylation FOXF1 gene Figures Figure 1 Figure 2 Background Colorectal cancer (CRC) is the third most frequent malignancy worldwide and one of the leading causes of cancer-related deaths. The burden of CRC is expected to rise to 60% by 2030 ( 1 , 2 ). In Iran, CRC ranks as the fourth most prevalent cancer ( 3 ), with its incidence expected to double by 2040. While CRC rates remain very low among the aging Iranian community, there is a concerning rise among younger Iranians ( 4 ). CRC develops as genetic and epigenetic changes gradually accumulate in precursor lesions such as adenomas and serrated lesions. These changes lead to the inactivation of tumor suppressor genes, chromosomal instability, and oncogenes activation( 5 , 6 ). CRC screening can remarkably improves tumor detection in the early stages ( 7 ). Colonoscopy remains the gold standard tool for CRC diagnosis; however, its invasiveness and high cost limit its use for population screening ( 8 , 9 ). The fecal occult blood test (FOBT) is a more affordable and noninvasive alternative, but results high of false-positive tests and require for frequent repetition( 10 ). Unfortunately, the majority of CRC patients are diagnosed at an advanced stage, underscoring the urgent requirement for the rapid identification of diagnostic and prognostic biomarkers ( 11 , 12 ). Colorectal cancer can potentially be identified through the detection of genomic biomarkers in stool or blood samples, with varying sensitivity levels ( 13 , 14 ). Biomarker-based screening offers significant advantages over classical methods such as imaging and endoscopy ( 15 ). There is now widespread consensus that methylation markers serve as reliable indicators for identifying early-stage CRC patients ( 16 ). One well-known epigenetic mechanism is the methylation of cytosine residues within CpG dinucleotide sequences, known as CpG islands, which are located in gene promoter regions. About 60% of human gene promoter regions contain CpG islands, mostly found at the 5' end of genes ( 17 ). Alterations in DNA methylation patterns are a hallmark of CRC and many other cancers. Studies have revealed that numerous genes exhibit hypermethylation in early-stage CRC, directly contributing to tumor development and progression. Thus, Various methylated genes may serve as valuable molecular markers for early tumor screening ( 18 , 19 ). Various studies have demonstrated that numerous members of the FOX family play an active role in cancer progression ( 20 ). FOXF1 is a forkhead box (FOX) transcription factor family member. Various FOX family members have been linked to various biological processes, including cell differentiation, development, cell cycle regulation, cell metabolism, aging, cell survival, immune responses, organogenesis, DNA repair, and embryonic development ( 21 – 23 ). Early research demonstrated that FOXF1 was found to be a factor in the progression of multiple cancers, such as osteosarcoma, breast cancer, lung cancer, and Colorectal cancer ( 24 – 28 ). Notably, FOXF1 acts as both an oncogene and a tumor suppressor in cancer development ( 21 ). Despite the increasing evidence pointing to FOXF1 's impact on cancer development, its potential as a biomarker for CRC detection in stool remains unexplored. In this study, we aimed to assess, for the first time, the potential of utilizing FOXF1 gene promoter methylation in stool samples as a noninvasive biomarker for distinguishing CRC patients from healthy controls. Furthermore, we compared its diagnostic sensitivity and specificity with FOBT to evaluate its potential as an alternative or complementary screening method. Materials and methods Sample Collection A total of 50 fecal specimens were obtained from CRC patients 7 days following colonoscopy and pathological confirmation of CRC. In addition, 50 stool samples were taken from healthy people 7 days after a colonoscopy confirmed their health. Consent forms were signed by all participants of the study. The studied population was made up of individuals who had been directed to Imam Reza Hospital situated in Kermanshah, Iran. The study was approved by the ethics committee of Kermanshah University of Medical Sciences, Iran (Ethics code: no 95285). DNA Extraction DNA was extracted from feces samples according to the manufacturer's instructions using the Gene All kit (South Korea, Cat number 115–150). In brief, 200 mg of stool sample and 1mL of PBS buffer were homogenized in a 2 mL microtube, the microtube was maintained at room temperature for several minutes, and the supernatant was separated to a 2 mL microtube, centrifuged at 13000 RPM for 2 min, and the supernatant was discarded. Then 1.3 mL of lysing buffer was added to the pellet and mixed. The tube was incubated at room temperature and then centrifuged at 12000 rpm for 5 min. The supernatant was transferred to an EZ pass filter and centrifuged at 12000 rpm for 1 min. The filter was placed on a 1.5 mL microtube, and 100 µL of elution buffer (EB) was added to the filter and centrifuged at 12000 rpm for 5 min. We added 500 µL of PB to the microtube's contents, transferred them to a G-type spin column, and centrifuged them for 1 min at 12000 rpm. 500 µL of washing buffer was added to the spin column and centrifuged at 12000 rpm for 1 min. We then added 50 µL of EB buffer to the column and centrifuged at 13000 rpm for 1 min and collected the DNA-containing solution in a new 1.5 mL microtube. The concentration of the extracted DNA was defined using a NanoDrop ND‑1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). DNA concentration was ranging between 20–100 ng/µl. Bisulfite Treatment DNA bisulfite treatment was performed to prepare DNA samples for Methylight PCR using the EpiTect bisulfite kit in conformity with the industrialist’s protocol (Qiagen, Germany). The EpiTect bisulfite kit was used to modify 20–40µl of DNA. DNA incubation with high concentrations of sodium bisulfite salt at low pH and high temperature leads to a change in unmethylated cytosine residues into uracil, leaving the methylcytosine unchanged. Primer and Probe Design primers and a TaqMan probe were constructed using Beacon Designer™ (version 8.13; http://www.premierbiosoft.com/molecular_beacons/ ) to specifically anneal full-methylated DNA at the FOXF1 promoter gene. The ALU-C4 sequences were used as a reference control. All primers and probes were synthesized by Metabion Biotechnology Company (Metabion, steinkirchen, Germany) (Table 1 ). Table 1 Primer and probe sequences applied for MethyLight PCR. MethyLight, a TaqMan-based Methylation-Specific Real-time PCR technique MethyLight PCR was carried out by an Applied Biosystems (ABI) 7500 Real-time device (Foster City, CA, USA), using the EpiTect MethyLight PCR + ROX Vial Kit (Qiagen, Germany) according to the manufacturer’s protocol: Reactions were done in 20 µl volume including 10 µl of 1XEpiTect Methylation MasterMix, 5 µl mix including of 0.4 µM forward and reverse primer and 0.2 µM TaqMan probe, 50x ROX Dye Solution 0.4 µl, 3–4 µl RNase –free water and 1–2 µl converted DNA. Thermal cycling was done in 45 cycles: 5 min at 95ᵒC for the initial PCR activation step, 15 s at 95ᵒC for denaturation, and 60 s at 60ᵒC for the annealing and extension step Each plate consisted of 1 µl from patient DNA, full-methylated bisulfite transformed DNA as a positive control (EpiTect PCR Control DNA, Qiagen, Germany), unmethylated human bisulfite-converted DNA (EpiTect PCR Control DNA, Qiagen, Germany,), and RNase free water, used as a negative control with each set of primers and probes. The standard curve was plotted using the Ct value of commercial methylated control DNA (EpiTect PCR Control DNA, Qiagen, Germany Cat.N. 59655) with different concentrations versus the log value of input concentration DNA. We acquired an acceptable methylight PCR result, which is shown by the PCR efficiency (92–105%) and correct slope value (-3.65 up to − 3.2). The standard curve was obtained by plotting the Ct values of methylated control DNA as a function of DNA concentrations. FOBT An alcohol pyrimidine-based FOBT was performed to assess the presence of blood in all of the stool samples. For this, we suspended a spatula of the stool in normal saline in a microtube and mixed it. Pyramidone alcohol and 4–5 drops of 10% acetic acid were added and mixed. 5–6 drops of 10% H2O2 were added as a substrate. Pyrimidine alcohol oxidizes the oxygen released from H2O2 decomposition by the peroxidase enzyme, causing a color change. The color change to purple indicates the presence of blood in the medium. The activity of the peroxidase enzyme or blood content in the stool determines the extent of color change intensity. Data Analysis The PCR data was evaluated by ABI 7500 SDS version 1.3.1 software-USA. For all samples, the percentage of the methylation reference (PMR) was determined using the formula: [( FOXF1 /ALU) sample/( FOXF1 /ALU) positive control]x100. Statistical data analysis was done using SPSS software (version 16.0, IBM-USA). P-value < 0.05 was considered to be significantly acceptable. To evaluate the sensitivity and specificity of the methylation level of the FOXF1 , ROC analysis was defined. Results Demographic results Overall, 50 stool samples were obtained from patients with CRC and 50 samples from the control group. The average age of the patients was 56±12.9 years. the healthy controls were matched with the patient group in terms of age and sex. There was no obvious difference in age or gender between CRC patients and control groups (Table 2). Table 2 The demographic characteristics of patients group with CRC and healthy control. Demographic features Patient group (number=50) control group (number=50) P value* Age(years) Gender (Female: Male) 56±12.9 3:2 54 ± 12 9:7 0.166 0.686 *Mann-Whitney U Test FOXF1 gene promoter methylation Median PMR values of stool samples from patients and healthy controls were 0.06 (95%CI 0.01-13.1) and 0.01 (95% CI 0.0-0.02) respectively (Table 3 and Figure 1). In stool samples from CRC patients, PMR values were higher than those of the control group (P<0.001). PMR values showed no significant difference in demographic or pathological features between the cohorts used in the study (Table 4). The highest value of PMR in the stool samples from healthy controls was considered a threshold of methylation status, while a PMR value higher than that was considered a positive methylation status. Methylation of the FOXF1 gene (positive results) in the age group >50 years was compared to the age group ≤50 (73.5 % vs. 75%) and also between female patients compared to males (80% vs. 65%), (Table 5). There was no major difference in the level of methylation between the gender and age sub-groups (Table 5). The ability of FOXF1 gene PMR level as a biomarker Furthermore, we utilized the ROC curve to assess how well the FOXF1 gene PMR level in stool samples for distinguishing between CRC patients and controls. The examination of the ROC curve analysis showed that the sensitivity and specificity of the FOXF1 gene at the optimal cut-off value with an AUC of 0.744 (95% CI: P<0.001) were 59% and 96%, respectively (Fig 2). Table 3 Median PMR value results for FOXF1 gene promoter methylation in the stool samples of patients with CRC and healthy control. P-value* Percentiles** Maximum Minimum No. of patients 75th 50th 25th <0.001 13.1 0.02 0.06 0.01 0.01 0.0 73 0.03 0.0 0.0 50 50 Patient group Normal group Stool PMR *Mann–Whitney U test **Explore Table 4 Association of stool PMR values (percentiles) for FOXF1 gene with demographic and pathological characteristics of the patient group. P value* Stool PMR (Percentiles)** number Characteristics 75th 50th 25th 0.5 0.41 0.97 0.06 0.31 0.13 13.37 13.6 10.87 14.95 9.15 14.5 5.62 20.25 9.02 17.25 10.1 15.6 2.64 3.48 3.9 2.55 3.1 2.85 2.5 4.6 2.65 4.7 3.65 4.1 0.99 2.05 2.07 1.1 1.91 1.98 1.34 2.04 1.91 1.74 2.2 2.71 16 34 30 20 27 23 21 29 25 25 28 22 Age ≤ 50 years >50 years Gender Female Male Grade Poorly+Moderately Well Location Distal Proximal Size ≤10mm >10mm Stage Stage I & II Stage III & IV *Mann-Whitney U Test **Explore Table 5 Association Between FOXF1 gene Promoter Hypermethylation and Clinicopathological Characteristics P value* hypermethylation status** No. of patients Characteristics Negative(%) Positive (%) 0.61 0.14 0.53 0.74 0.34 0.15 <0.001 4(25%) 9(26.5%) 6(20%) 7(35%) 2(20%) 11(27.5%) 6(28.6%) 7(24.1%) 8(32%) 5(20%) 5(17.9%) 3(16.7%) - - 12(75%) 25(73.5%) 24(80%) 13(65%) 8(80%) 29(72.5%) 15(71.4%) 22(75.9%) 17(68%) 20(80%) 23(82.1%) 19(83.3%) 0.06 (95%CI 0.01-13.1) 0.01 (95%CI: 0.0-0.02) 16 34 30 20 27 23 21 29 25 25 28 22 Age ≤ 50 years >50 years Gender Female Male Histological Grade Poorly+Moderately Well Tumor location Distal Proximal Tumor size ≤10mm >10mm Tumor Stage Stage I & II Stage III & IV Methylation Levels (PMR Values) Median PMR (CRC Patients) Median PMR (Healthy Controls) * Fisherʼs Exact Test **Crosstabs FOBT results We also examined the FOBT as an early diagnostic test. The sensitivity of FOBT was 38% compared to 59% for FOXF1 gene promoter methylation. These results showed that the sensitivity of FOXF1 gene promoter methylation in stool samples was significantly higher than that of the FOBT. Discussion Colorectal cancer is a major public health concern, accounting for about 10% of worldwide incidence and mortalities ( 29 ). In the development of CRC, there is a gradual accumulation of different genetic and epigenetic changes, with abnormal DNA methylation being a crucial mechanism ( 30 ). Aberrant DNA methylation patterns are commonly observed in gastrointestinal neoplasms and could serve as valuable markers for screening, diagnosis, prognosis determination, and prediction of CRC. In addition, the plentiful presence in extracellular environments, resistance to degradation, and presence in plasma, serum, feces specimens, and other noninvasive bodily fluids facilitates quantitative analysis of methylated DNA biomarkers ( 31 ). Collecting bodily fluids is a non-intrusive process that allows for the early identification of cancer or disease monitoring ( 32 ). Numerous studies have validated stool-based methylated DNA detection as an effective method for early CRC screening ( 31 ). Promoter hypermethylation contributes to the inactivation of tumor suppressor genes and ultimately oncogenic processes by affecting key cellular pathways ( 16 , 33 ). Common methylated and silenced genes often seen in CRC include vimentin(VIM), cadherin-1(CDH1), MLH1, TIMP metallopeptidase inhibitor-3 (TIMP3), hypermethylated in cancer-1 (HIC1) and secreted frizzled related protein-1(SFRP1) ( 33 ). Recently, methylated syndecan-2 (mSDC2) and methylated SEPT9 (mSEPT9) have identified as promising biomarkers for non-invasive detection of CRC ( 34 – 37 ). Researchers have studied abnormal methylation of different genes in the tissues and bodily fluids of individuals with CRC, which could be used as valuable biomarkers in screening for CRC ( 16 ).Cologuard is the first multitarget stool DNA test that has received FDA approval for CRC screening. It evaluates KRAS mutations, as well as BMP3 and NDRG4 methylation levels ( 13 , 38 ). Moreover, NDRG4, BMP3, and SEPT9 are now incorporated as DNA methylation markers in FDA-approved CRC screening tests ( 39 ). Therefore, we aimed to identify non-invasive biomarkers capable of detecting CRC in small stool samples. Analyzing single genes in epigenetic studies could provide a more cost-effective option for screening. Nevertheless, these studies have presented remarkable initial findings about these single epigenetic markers and their utility as stool DNA–based assays ( 40 ). Moradi et al. reported that PMR values in the stool of CRC patients was higher than those of healthy controls. Also, the analysis of SOX21 gene promoter methylation using a stool-based assay shows a notably high level of sensitivity (72.5%) and specificity (100%) ( 41 ). Zhang et al. showed that SPG20 promoter hypermethylation serves as a highly sensitive and specific biomarker in the detection of CRC using stool samples (80.2% and 100%, respectively) ( 42 ). Glöckner et al. in stage I to III CRC patients, TFPI2 methylation was found in stool DNA with a sensitivity ranging from 76–89% and a specificity ranging from 79–93%. recognition of TFPI2 methylation in fecal DNA may serve as a biomarker for CRC screening ( 40 ).Muller et al. identified a sensitivity of 77–90% and a specificity of 77% for SFRP2 methylation as a sensitive single DNA-based biomarker for detecting CRC in stool samples from 49 patients ( 43 ). According to research single marker methylation-based stool using Vimentin and SFRP2, the sensitivity has found to be 52% and 87–94%, while the specificity has reported 90% and 85–90%, respectively ( 44 – 47 ). Various studies have indicated that the FOX family plays a crucial role in cellular proliferation, apoptosis, metastasis, the cell cycle, cellular metabolism, progression, and cancer biology in various cancers ( 48 – 51 ). In total, Dysregulation of FOX proteins may impact the cell fate, tumor growth, and metastasis of cancer ( 52 ). FOXF1 , a transcription factor, attaches to the promoter regions of target genes to control gene expression levels and influence the function of various signaling pathways crucial for physiological and pathological processes ( 20 ). FOXF1 is critical for controlling angiogenesis during embryonic development and may also be involved in angiogenesis, impacting CRC progression. Increased FOXF1 expression has been linked to aggressive CRC features, including poor differentiation and advanced disease stages. There was a significant association between increased FOXF1 expression and poorer prognosis in CRC patients ( 28 ). In contrast, FOXF1 expression is often silenced through DNA hypermethylation in breast cancer, where it acts as a tumor suppressor ( 53 ). Herrera-Merchan et al. reported that FOXF1 expression could also be silenced via promoter methylation in lung adenocarcinoma and functions as a tumor suppressor ( 54 ). In another study, Mitchell et al. analyzed a panel of 23 genes in the blood and tissue of CRC patients compared to a control group. They found increased DNA methylation in at least half of CRC tissue samples compared to normal tissue. Additionally, 11 genes, including FOXF1 , exhibited low methylation levels in peripheral blood DNA from healthy individuals ( 55 ). On the other hand, FOXF1 promotes the development and maintenance of gastrointestinal stromal tumors through the regulation of KIT and ETV1 expression ( 56 ). In hepatocellular carcinoma, FOXF1 serves as a suppressor of cell invasion and tumorigenesis, with its expression closely linked to patient survival rates ( 57 ). Furthermore, in CRC cell lines with p53 inactivation, FOXF1 expression is silenced, leading to genomic instability in FOXF1 -expressing CRC cells with a dysfunctional p53-p21WAF1 checkpoint. This underscores FOXF1' s significance in CRC development ( 58 ). Additionally, the competitive binding of miR-375-3p by LINC00022 could lead to the up-regulation of FOXF1 , potentially contributing to CRC progression. ( 59 ). Lo et al. reported cytoplasmic mislocalization of upregulated FOXF1 protein in CRC epithelial cells, which is associated with poor prognosis factors ( 21 ). Furthermore, FOXF1 upregulation is related to poorer diagnosis, the advanced progression, invasion, and metastasis of CRC ( 20 ). Recently reported that FOXF1 gene methylation levels in the plasma of CRC patients are higher than healthy controls ( 60 ). In the present study, for the first time, we determined the FOXF1 gene promoter methylation for 50 stool samples patients with CRC and 50 healthy controls. Our results showed that The median PMR values of stool samples from patients and healthy controls were between 0.01 to 13.1 and 0.0 to 0.02, respectively. In addition, we examined the association of the PMR values obtained from stool samples with the demographic and pathologic features of the patients. There was no statistically significant correlation found between the methylation levels of the FOXF1 gene promoter and age, gender, histological grade, tumor stage, tumor location, or tumor size in CRC patients. In our research, FOXF1 gene promoter methylation position was assessed by the MethyLight technique, a quantitative method with high sensitivity and specificity capable of assaying a small amount of DNA. Our data revealed that the FOXF1 gene promoter methylation had higher sensitivity and specificity than FOBT in stool samples. Although our results offer valuable information into the potential of FOXF1 methylation as a noninvasive biomarker for CRC detection in stool samples, certain limitations must be considered. the sample size is a relatively small and may affect the generalizability of our results. Furthermore, potential biases in patient selection and sample collection could impact the observed methylation patterns. To validate our findings and better evaluate the clinical utility of FOXF1 methylation in CRC detection, future studies should include larger, more diverse cohorts. Conclusion In summary, as the first research to evaluate FOXF1 gene promoter methylation in stool samples of patients with CRC, it can be concluded that the PMR value for FOXF1 in CRC patients is higher than in healthy controls. The quantification of FOXF1 gene promoter methylation in stool samples shows promise as a non-invasive biomarker for CRC screening, with a sensitivity of 59% and a specificity of 96%. These results suggest potential sensitivity advantages over FOBT. Additionally, FOXF1 gene promoter methylation was found to be independent of age, sex, tumor grade, or stage, highlighting its potential utility in diverse patient populations. Future research with larger cohorts and more diverse populations is necessary to confirm these findings and explore its feasibility as a cost-effective screening tool. Limitations The study has several limitations that must be addressed to strengthen its findings. One key limitation is the relatively small number of participants in the study, which may have reduced its statistical power. Moreover, the limited sample size could impact the generalizability of the results to a wider population Therefore, it is crucial to conduct further research with a larger sample size to enhance the validity and practicality of our results. Another limitation is that several important risk factors for colorectal cancer, such as smoking, diet, and alcohol consumption, were not investigated due to insufficient clinical data. Furthermore, the research was carried out at a lone center. So The homogeneity of the population and inadequate analysis of confounding factors may also affect the reliability of the results. Furthermore, focusing solely on FOXF1 methylation without exploring additional biomarkers reduces the potential diagnostic accuracy. Methodological constraints, such as the standardization of stool sample collection, and the absence of a cost-effectiveness analysis further limit the study's real-world applicability. Future work should address these issues through larger, more diverse cohorts and comprehensive validation studies. Methylation-based evaluation is susceptible to false-positive and false-negative results due to various biological and technical factors. For example, contamination of stool samples with residual methylated DNA from food or external sources can lead to false positives. Also, Minimal amounts of DNA may be shed into the stool in the early stages of the disease, which reduces the ability to detect methylated DNA and can lead to false-negative. Abbreviations CRC: Colorectal cancer FOXF1 : Forkhead box F1 FOBT: Fecal occult blood test PMR: Percentage of the methylated reference CIMP: CpG Island Methylator Phenotype Declarations Acknowledgments The authors are grateful to the staff at the Medical Genetics Laboratory, Reference Laboratory, and Kermanshah University of Medical Sciences for their support throughout this research. Authors’ contributions Bayan Mehrjoei󠅥 and Nayebali Rezvani designed the research. Susan Tahmasebi performed stool samples and clinical data collection; Nayebali Rezvani analyzed data; Bayan Mehrjoei󠅥 wrote the paper. Asad Vaisi-Raygani edited the final version of the manuscript. Source of Funding: No funding Conflict of interest: There are no conflicts of interest. Consent for publication: Not applicable. Ethical approval and consent to participate All individuals partaking were tasked with signing the informed consent form. 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Methylated SFRP2 and SDC2 in stool specimens for Colorectal Cancer early detection: A cost-effective strategy for Chinese population. J Cancer. 2021;12(9):2665. Song L, Jia J, Peng X, Xiao W, Li Y. The performance of the SEPT9 gene methylation assay and a comparison with other CRC screening tests: A meta-analysis. Sci Rep. 2017;7(1):3032. Ahlquist DA, Zou H, Domanico M, Mahoney DW, Yab TC, Taylor WR, et al. Next-generation stool DNA test accurately detects colorectal cancer and large adenomas. Gastroenterology. 2012;142(2):248–56. Koch A, Joosten SC, Feng Z, de Ruijter TC, Draht MX, Melotte V, et al. Analysis of DNA methylation in cancer: location revisited. Nat reviews Clin Oncol. 2018;15(7):459–66. Glöckner SC, Dhir M, Yi JM, McGarvey KE, Van Neste L, Louwagie J, et al. Methylation of TFPI2 in stool DNA: a potential novel biomarker for the detection of colorectal cancer. Cancer Res. 2009;69(11):4691–9. Moradi K, Babaei E, Feizi MAH, 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. 2021;58(2):217–24. Zhang H, Song Y-C, Dang C-X. Detection of hypermethylated spastic paraplegia-20 in stool samples of patients with colorectal cancer. Int J Med Sci. 2013;10(3):230. Müller HM, Oberwalder M, Fiegl H, Morandell M, Goebel G, Zitt M, et al. Methylation changes in faecal DNA: a marker for colorectal cancer screening? Lancet. 2004;363(9417):1283–5. Chen W-D, Han ZJ, Skoletsky J, Olson J, Sah J, Myeroff L, et al. Detection in fecal DNA of colon cancer–specific methylation of the nonexpressed vimentin gene. J Natl Cancer Inst. 2005;97(15):1124–32. Wang D-R, Tang D. Hypermethylated SFRP2 gene in fecal DNA is a high potential biomarker for colorectal cancer noninvasive screening. World J gastroenterology: WJG. 2008;14(4):524. Huang Z, Li L, Wang J. Hypermethylation of SFRP2 as a potential marker for stool-based detection of colorectal cancer and precancerous lesions. Dig Dis Sci. 2007;52:2287–91. Oberwalder M, Zitt M, Wöntner C, Fiegl H, Goebel G, Zitt M, et al. SFRP2 methylation in fecal DNA—a marker for colorectal polyps. Int J Colorectal Dis. 2008;23:15–9. Kim CG, Lee H, Gupta N, Ramachandran S, Kaushik I, Srivastava S, et al. editors. Role of Forkhead Box Class O proteins in cancer progression and metastasis. Seminars in cancer biology. Elsevier; 2018. Weng W, Okugawa Y, Toden S, Toiyama Y, Kusunoki M, Goel A. FOXM1 and FOXQ1 are promising prognostic biomarkers and novel targets of tumor-suppressive miR-342 in human colorectal cancer. Clin Cancer Res. 2016;22(19):4947–57. Cui Y-M, Jiang D, Zhang S-H, Wu P, Ye Y-P, Chen C-M, et al. FOXC2 promotes colorectal cancer proliferation through inhibition of FOXO3a and activation of MAPK and AKT signaling pathways. Cancer Lett. 2014;353(1):87–94. Xie R, Wang J, Liu X, Wu L, Zhang H, Tang W, et al. RUFY3 interaction with FOXK1 promotes invasion and metastasis in colorectal cancer. Sci Rep. 2017;7(1):3709. Myatt SS, Lam EW-F. The emerging roles of forkhead box (Fox) proteins in cancer. Nat Rev Cancer. 2007;7(11):847–59. Lo P-K, Lee JS, Liang X, Han L, Mori T, Fackler MJ, et al. Epigenetic inactivation of the potential tumor suppressor gene FOXF1 in breast cancer. Cancer Res. 2010;70(14):6047–58. Herrera-Merchan A, Cuadros M, Rodriguez MI, Rodriguez S, Torres R, Estecio M, et al. The value of lncRNA FENDRR and FOXF1 as a prognostic factor for survival of lung adenocarcinoma. Oncotarget. 2017;11(13):1172. Mitchell SM, Ross JP, Drew HR, Ho T, Brown GS, Saunders NF, et al. A panel of genes methylated with high frequency in colorectal cancer. BMC Cancer. 2014;14:1–15. Ran L, Chen Y, Sher J, Wong EW, Murphy D, Zhang JQ, et al. FOXF1 defines the core-regulatory circuitry in gastrointestinal stromal tumor. Cancer Discov. 2018;8(2):234–51. Zhao Z-g, Wang D-q, Hu D-f, Li Y-s, Liu S-h. Decreased FOXF1 promotes hepatocellular carcinoma tumorigenesis, invasion, and stemness and is associated with poor clinical outcome. OncoTargets therapy. 2016:1743–52. Lo P-K, Lee JS, Sukumar S. The p53–p21WAF1 checkpoint pathway plays a protective role in preventing DNA rereplication induced by abrogation of FOXF1 function. Cell Signal. 2012;24(1):316–24. Xu L, He H, Shang Y, Qu X, Sun J. LINC00022 acts as an oncogene in colorectal cancer progression via sponging miR-375-3p to regulate FOXF1 expression. BMC Cancer. 2022;22(1):453. Dastafkan Z, Rezvani N, Amini S. Diagnostic value of FOXF1 gene promoter-methylated DNA in the plasma samples of patients with colorectal cancer. Int J Biol Mark. 2023;38(3–4):194–202. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5551296","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":431273334,"identity":"da061bc1-652e-4f10-b560-16327bebdee7","order_by":0,"name":"Susan Tahmasebi","email":"","orcid":"","institution":"Kermanshah University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Susan","middleName":"","lastName":"Tahmasebi","suffix":""},{"id":431273335,"identity":"ddd15e6a-c75d-4ec1-9858-6a4630b15b71","order_by":1,"name":"Bayan Mehrjoei","email":"","orcid":"","institution":"Kermanshah University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Bayan","middleName":"","lastName":"Mehrjoei","suffix":""},{"id":431273336,"identity":"2787283c-e2d1-4c62-af8c-9d347108d563","order_by":2,"name":"Asad Vaisi-Raygani","email":"","orcid":"","institution":"Kermanshah University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Asad","middleName":"","lastName":"Vaisi-Raygani","suffix":""},{"id":431273337,"identity":"cd485359-2244-4e94-9051-2bb12e30f018","order_by":3,"name":"Nayebali Rezvani","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3klEQVRIie2PPQrCQBCFVxY2zZNtVxC9wkrAHyy8SmxSxRuICAFLa8FLWFkrg6kWawu7gIVVJGApJlZWce0E9ytmmvcx8xhzOH4YjGK+K7Zq2ivaE0GpwP6MltAv92NSrolyTM/NHkd+PU37YB4dNlWKOoaBj+SCQVzfDqOkeAxheKo8Y6DHK0FgVN/6kSgUhW6l0jYy268epYKLHz0sFG1Qm98WBE3g6WRhoXSM8NltSWjEossnSwXxqUvL8DQL7jSSktI8us9a0qOkuv4bQr2mbbyEZ9+kHQ6H4394AjxpQrWq6dUQAAAAAElFTkSuQmCC","orcid":"","institution":"Kermanshah University of Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Nayebali","middleName":"","lastName":"Rezvani","suffix":""}],"badges":[],"createdAt":"2024-11-29 20:23:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5551296/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5551296/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":78978450,"identity":"e5657c04-20c8-4b58-a93a-502debaa3eff","added_by":"auto","created_at":"2025-03-21 15:41:12","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":5806,"visible":true,"origin":"","legend":"\u003cp\u003eBox plot for \u003cem\u003eFOXF1\u003c/em\u003e gene PMR value of stool samples in case and control groups. The boxes show the interquartile range, (interval between the 25th and 75th percentile). The lines inner the boxes indicate median values.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5551296/v1/665d9d249d7e0e9d69228892.png"},{"id":78976883,"identity":"45926a3f-f446-4da0-9e43-5d6e157c086c","added_by":"auto","created_at":"2025-03-21 15:25:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":13213,"visible":true,"origin":"","legend":"\u003cp\u003eROC curve for \u003cem\u003eFOXF1\u003c/em\u003e gene promoter methylation in stool samples.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5551296/v1/a50eade4ad8c67a37b77dc4c.png"},{"id":79912032,"identity":"df766b75-17fc-4536-8095-b8532ec891cf","added_by":"auto","created_at":"2025-04-04 11:47:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":911972,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5551296/v1/1d324f3e-3e77-4c78-b0ab-d0163e27b7aa.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Evaluation of FOXF1 promoter DNA methylation, as a promising epigenetic biomarker for colorectal cancer in stool samples","fulltext":[{"header":"Background","content":"\u003cp\u003eColorectal cancer (CRC) is the third most frequent malignancy worldwide and one of the leading causes of cancer-related deaths. The burden of CRC is expected to rise to 60% by 2030 (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). In Iran, CRC ranks as the fourth most prevalent cancer (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e), with its incidence expected to double by 2040. While CRC rates remain very low among the aging Iranian community, there is a concerning rise among younger Iranians (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). CRC develops as genetic and epigenetic changes gradually accumulate in precursor lesions such as adenomas and serrated lesions. These changes lead to the inactivation of tumor suppressor genes, chromosomal instability, and oncogenes activation(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). CRC screening can remarkably improves tumor detection in the early stages (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Colonoscopy remains the gold standard tool for CRC diagnosis; however, its invasiveness and high cost limit its use for population screening (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). The fecal occult blood test (FOBT) is a more affordable and noninvasive alternative, but results high of false-positive tests and require for frequent repetition(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Unfortunately, the majority of CRC patients are diagnosed at an advanced stage,\u003c/p\u003e \u003cp\u003eunderscoring the urgent requirement for the rapid identification of diagnostic and prognostic biomarkers (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eColorectal cancer can potentially be identified through the detection of genomic biomarkers in stool or blood samples, with varying sensitivity levels (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Biomarker-based screening offers significant advantages over classical methods such as imaging and endoscopy (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). There is now widespread consensus that methylation markers serve as reliable indicators for identifying early-stage CRC patients (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). One well-known epigenetic mechanism is the methylation of cytosine residues within CpG dinucleotide sequences, known as CpG islands, which are located in gene promoter regions. About 60% of human gene promoter regions contain CpG islands, mostly found at the 5' end of genes (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Alterations in DNA methylation patterns are a hallmark of CRC and many other cancers. Studies have revealed that numerous genes exhibit hypermethylation in early-stage CRC, directly contributing to tumor development and progression. Thus, Various methylated genes may serve as valuable molecular markers for early tumor screening (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eVarious studies have demonstrated that numerous members of the FOX family play an active role in cancer progression (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). \u003cem\u003eFOXF1\u003c/em\u003e is a forkhead box (FOX) transcription factor family member. Various FOX family members have been linked to various biological processes, including cell differentiation, development, cell cycle regulation, cell metabolism, aging, cell survival, immune responses, organogenesis, DNA repair, and embryonic development (\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Early research demonstrated that \u003cem\u003eFOXF1\u003c/em\u003e was found to be a factor in the progression of multiple cancers, such as osteosarcoma, breast cancer, lung cancer, and Colorectal cancer (\u003cspan additionalcitationids=\"CR25 CR26 CR27\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Notably, \u003cem\u003eFOXF1\u003c/em\u003e acts as both an oncogene and a tumor suppressor in cancer development (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDespite the increasing evidence pointing to \u003cem\u003eFOXF1\u003c/em\u003e's impact on cancer development, its potential as a biomarker for CRC detection in stool remains unexplored. In this study, we aimed to assess, for the first time, the potential of utilizing \u003cem\u003eFOXF1\u003c/em\u003e gene promoter methylation in stool samples as a noninvasive biomarker for distinguishing CRC patients from healthy controls. Furthermore, we compared its diagnostic sensitivity and specificity with FOBT to evaluate its potential as an alternative or complementary screening method.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eSample Collection\u003c/h2\u003e\n \u003cp\u003eA total of 50 fecal specimens were obtained from CRC patients 7 days following colonoscopy and pathological confirmation of CRC. In addition, 50 stool samples were taken from healthy people 7 days after a colonoscopy confirmed their health. Consent forms were signed by all participants of the study. The studied population was made up of individuals who had been directed to Imam Reza Hospital situated in Kermanshah, Iran. The study was approved by the ethics committee of Kermanshah University of Medical Sciences, Iran (Ethics code: no 95285).\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eDNA Extraction\u003c/h3\u003e\n\u003cp\u003eDNA was extracted from feces samples according to the manufacturer\u0026apos;s instructions using the Gene All kit (South Korea, Cat number 115\u0026ndash;150). In brief, 200 mg of stool sample and 1mL of PBS buffer were homogenized in a 2 mL microtube, the microtube was maintained at room temperature for several minutes, and the supernatant was separated to a 2 mL microtube, centrifuged at 13000 RPM for 2 min, and the supernatant was discarded. Then 1.3 mL of lysing buffer was added to the pellet and mixed. The tube was incubated at room temperature and then centrifuged at 12000 rpm for 5 min. The supernatant was transferred to an EZ pass filter and centrifuged at 12000 rpm for 1 min. The filter was placed on a 1.5 mL microtube, and 100 \u0026micro;L of elution buffer (EB) was added to the filter and centrifuged at 12000 rpm for 5 min. We added 500 \u0026micro;L of PB to the microtube\u0026apos;s contents, transferred them to a G-type spin column, and centrifuged them for 1 min at 12000 rpm. 500 \u0026micro;L of washing buffer was added to the spin column and centrifuged at 12000 rpm for 1 min. We then added 50 \u0026micro;L of EB buffer to the column and centrifuged at 13000 rpm for 1 min and collected the DNA-containing solution in a new 1.5 mL microtube. The concentration of the extracted DNA was defined using a NanoDrop ND‑1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). DNA concentration was ranging between 20\u0026ndash;100 ng/\u0026micro;l.\u003c/p\u003e\n\u003ch3\u003eBisulfite Treatment\u003c/h3\u003e\n\u003cp\u003eDNA bisulfite treatment was performed to prepare DNA samples for Methylight PCR using the EpiTect bisulfite kit in conformity with the industrialist\u0026rsquo;s protocol (Qiagen, Germany). The EpiTect bisulfite kit was used to modify 20\u0026ndash;40\u0026micro;l of DNA. DNA incubation with high concentrations of sodium bisulfite salt at low pH and high temperature leads to a change in unmethylated cytosine residues into uracil, leaving the methylcytosine unchanged.\u003c/p\u003e\n\u003ch3\u003ePrimer and Probe Design\u003c/h3\u003e\n\u003cp\u003eprimers and a TaqMan probe were constructed using Beacon Designer\u0026trade; (version 8.13; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.premierbiosoft.com/molecular_beacons/\u003c/span\u003e\u003c/span\u003e) to specifically anneal full-methylated DNA at the \u003cem\u003eFOXF1\u003c/em\u003e promoter gene. The ALU-C4 sequences were used as a reference control. All primers and probes were synthesized by Metabion Biotechnology Company (Metabion, steinkirchen, Germany) (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003cdiv align=\"left\" class=\"colspec\"\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e Primer and probe sequences applied for MethyLight PCR.\u003c/div\u003e\n \u003cp\u003e\u003cimg 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\"\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eMethyLight, a TaqMan-based Methylation-Specific Real-time PCR technique\u003c/h3\u003e\n\u003cp\u003eMethyLight PCR was carried out by an Applied Biosystems (ABI) 7500 Real-time device (Foster City, CA, USA), using the EpiTect MethyLight PCR\u0026thinsp;+\u0026thinsp;ROX Vial Kit (Qiagen, Germany) according to the manufacturer\u0026rsquo;s protocol: Reactions were done in 20 \u0026micro;l volume including 10 \u0026micro;l of 1XEpiTect Methylation MasterMix, 5 \u0026micro;l mix including of 0.4 \u0026micro;M forward and reverse primer and 0.2 \u0026micro;M TaqMan probe, 50x ROX Dye Solution 0.4 \u0026micro;l, 3\u0026ndash;4 \u0026micro;l RNase \u0026ndash;free water and 1\u0026ndash;2 \u0026micro;l converted DNA. Thermal cycling was done in 45 cycles: 5 min at 95ᵒC for the initial PCR activation step, 15 s at 95ᵒC for denaturation, and 60 s at 60ᵒC for the annealing and extension step Each plate consisted of 1 \u0026micro;l from patient DNA, full-methylated bisulfite transformed DNA as a positive control (EpiTect PCR Control DNA, Qiagen, Germany), unmethylated human bisulfite-converted DNA (EpiTect PCR Control DNA, Qiagen, Germany,), and RNase free water, used as a negative control with each set of primers and probes. The standard curve was plotted using the Ct value of commercial methylated control DNA (EpiTect PCR Control DNA, Qiagen, Germany Cat.N. 59655) with different concentrations versus the log value of input concentration DNA. We acquired an acceptable methylight PCR result, which is shown by the PCR efficiency (92\u0026ndash;105%) and correct slope value (-3.65 up to \u0026minus;\u0026thinsp;3.2). The standard curve was obtained by plotting the Ct values of methylated control DNA as a function of DNA concentrations.\u003c/p\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eFOBT\u003c/h2\u003e\n \u003cp\u003eAn alcohol pyrimidine-based FOBT was performed to assess the presence of blood in all of the stool samples. For this, we suspended a spatula of the stool in normal saline in a microtube and mixed it. Pyramidone alcohol and 4\u0026ndash;5 drops of 10% acetic acid were added and mixed. 5\u0026ndash;6 drops of 10% H2O2 were added as a substrate. Pyrimidine alcohol oxidizes the oxygen released from H2O2 decomposition by the peroxidase enzyme, causing a color change. The color change to purple indicates the presence of blood in the medium. The activity of the peroxidase enzyme or blood content in the stool determines the extent of color change intensity.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003eData Analysis\u003c/h2\u003e\n \u003cp\u003eThe PCR data was evaluated by ABI 7500 SDS version 1.3.1 software-USA. For all samples, the percentage of the methylation reference (PMR) was determined using the formula: [(\u003cem\u003eFOXF1\u003c/em\u003e/ALU) sample/(\u003cem\u003eFOXF1\u003c/em\u003e/ALU) positive control]x100. Statistical data analysis was done using SPSS software (version 16.0, IBM-USA). P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to be significantly acceptable. To evaluate the sensitivity and specificity of the methylation level of the \u003cem\u003eFOXF1\u003c/em\u003e, ROC analysis was defined.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eDemographic results\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOverall, 50 stool samples were obtained from patients with CRC and 50 samples from the control group. The average age of the patients was 56\u0026plusmn;12.9 years. the healthy controls were matched with the patient group in terms of age and sex. There was no obvious difference in age or gender between CRC patients and control groups (Table 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e The demographic characteristics of patients group with CRC and healthy control.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"600\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 21.4642%;\"\u003e\n \u003cp\u003eDemographic features\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.6306%;\"\u003e\n \u003cp\u003ePatient group\u003c/p\u003e\n \u003cp\u003e(number=50)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.6306%;\"\u003e\n \u003cp\u003econtrol group\u003c/p\u003e\n \u003cp\u003e(number=50)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 35.2745%;\"\u003e\n \u003cp\u003eP value*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 21.4642%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAge(years)\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eGender\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(Female: Male)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.6306%;\"\u003e\n \u003cp\u003e\u003cspan dir=\"RTL\"\u003e\u0026nbsp; 56\u0026plusmn;12.9\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan dir=\"RTL\"\u003e\u0026nbsp; \u0026nbsp; 3:2\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.6306%;\"\u003e\n \u003cp\u003e54 \u0026plusmn; 12\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan dir=\"RTL\"\u003e\u0026nbsp; \u0026nbsp;9:7\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 35.2745%;\"\u003e\n \u003cp\u003e0.166\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e0.686\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e*Mann-Whitney U Test\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFOXF1\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;gene promoter methylation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMedian PMR values of stool samples from patients and healthy controls were 0.06 (95%CI 0.01-13.1) and 0.01 (95% CI 0.0-0.02) respectively (Table 3 and Figure 1). In stool samples from CRC patients, PMR values were higher than those of the control group (P\u0026lt;0.001). PMR values showed no significant difference in demographic or pathological features between the cohorts used in the study (Table 4). The highest value of PMR in the stool samples from healthy controls was considered a threshold of methylation status, while a PMR value higher than that was considered a positive methylation status. Methylation of the \u003cem\u003eFOXF1\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/em\u003egene (positive results) in the age group \u0026gt;50 years was compared to the age group \u0026le;50 (73.5 % vs. 75%) and also between female patients compared to males (80% vs. 65%), (Table 5). There was no major difference in the level of methylation between the gender and age sub-groups (Table 5).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe ability of \u003cem\u003eFOXF1\u003c/em\u003e gene PMR level as a biomarker\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFurthermore, we utilized the ROC curve to assess how well the \u003cem\u003eFOXF1\u003c/em\u003e gene PMR level in stool samples for distinguishing between CRC patients and controls. The examination of the ROC curve analysis showed that the sensitivity and specificity of the \u003cem\u003eFOXF1\u003c/em\u003e gene at the optimal cut-off value with an AUC of 0.744 (95% CI: P\u0026lt;0.001) were 59% and 96%, respectively (Fig 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3\u0026nbsp;\u003c/strong\u003eMedian PMR value results for \u003cem\u003eFOXF1\u003c/em\u003e gene promoter methylation in the stool samples of patients with CRC and healthy control.\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable dir=\"rtl\" border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"666\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp dir=\"LTR\"\u003eP-value*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp dir=\"LTR\"\u003e\u003cspan dir=\"RTL\"\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Percentiles**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp dir=\"LTR\"\u003eMaximum\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 91px;\"\u003e\n \u003cp dir=\"LTR\"\u003eMinimum\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp dir=\"LTR\"\u003eNo. of patients\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" rowspan=\"2\" valign=\"top\" style=\"width: 148px;\"\u003e\n \u003cp dir=\"LTR\"\u003e\u003cstrong\u003e\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp dir=\"LTR\"\u003e75th\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp dir=\"LTR\"\u003e\u003cspan dir=\"RTL\"\u003e\u0026nbsp; 50th\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp dir=\"LTR\"\u003e25th\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp dir=\"LTR\"\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp dir=\"LTR\"\u003e13.1\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp dir=\"LTR\"\u003e\u003cspan dir=\"RTL\"\u003e\u0026nbsp;0.06\u003c/span\u003e\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u003cspan dir=\"RTL\"\u003e\u0026nbsp;0.01\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp dir=\"LTR\"\u003e0.01\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp dir=\"LTR\"\u003e\u003cspan dir=\"RTL\"\u003e\u0026nbsp; 73\u003c/span\u003e\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u003cspan dir=\"RTL\"\u003e\u0026nbsp; 0.03 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 91px;\"\u003e\n \u003cp dir=\"LTR\"\u003e\u003cspan dir=\"RTL\"\u003e\u0026nbsp; \u0026nbsp; 0.0\u003c/span\u003e\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u003cspan dir=\"RTL\"\u003e\u0026nbsp; \u0026nbsp; 0.0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp dir=\"LTR\"\u003e\u003cspan dir=\"RTL\"\u003e\u0026nbsp;50\u003c/span\u003e\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u003cspan dir=\"RTL\"\u003e\u0026nbsp;50\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp dir=\"LTR\"\u003ePatient group\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003eNormal group\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 51px;\"\u003e\n \u003cp dir=\"LTR\"\u003e\u003cstrong\u003eStool PMR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e*Mann\u0026ndash;Whitney U test\u003c/p\u003e\n\u003cp\u003e**Explore\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4\u003c/strong\u003e Association of stool PMR values (percentiles) for \u003cem\u003eFOXF1\u003c/em\u003e gene with demographic and pathological characteristics of the patient group.\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable dir=\"rtl\" border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"628\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp dir=\"LTR\"\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp;P value*\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 252px;\"\u003e\n \u003cp dir=\"LTR\"\u003e\u003cstrong\u003eStool PMR (Percentiles)**\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp dir=\"LTR\"\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;number\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp dir=\"LTR\"\u003e\u003cstrong\u003eCharacteristics\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp dir=\"LTR\"\u003e\u003cstrong\u003e75th\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp dir=\"LTR\"\u003e\u003cstrong\u003e50th\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp dir=\"LTR\"\u003e\u003cstrong\u003e25th\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp dir=\"LTR\"\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e0.5\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;0.41\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp; 0.97\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; 0.06\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;0.31\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;0.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e13.37\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e13.6\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;10.87\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e14.95\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e9.15\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp; \u0026nbsp; 14.5\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp; \u0026nbsp;5.62\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e20.25\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; 9.02\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e17.25\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e10.1\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e15.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e2.64\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e3.48\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;3.9\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e2.55\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e3.1\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;2.85\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;2.5\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e4.6\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; 2.65\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e4.7\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e3.65\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e4.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u003cspan dir=\"RTL\"\u003e0.99\u003c/span\u003e\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e2.05\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; 2.07\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e1.1\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e1.91\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; 1.98\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; 1.34\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e2.04\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; 1.91\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e1.74\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e2.2\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e2.71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e16\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e34\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;30\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e20\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e27\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;23\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;21\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e29\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; 25\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; 25\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; 28\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; 22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp dir=\"LTR\"\u003e\u003cstrong\u003eAge\u003c/strong\u003e\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026le; 50 years\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026gt;50 years\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u003cstrong\u003eGender\u003c/strong\u003e\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003eFemale\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003eMale\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u003cstrong\u003eGrade\u003c/strong\u003e\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003ePoorly+Moderately\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003eWell\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u003cstrong\u003eLocation\u003c/strong\u003e\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003eDistal\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003eProximal\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u003cstrong\u003eSize\u003c/strong\u003e\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026le;10mm\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u0026gt;10mm\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u003cstrong\u003eStage\u003c/strong\u003e\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003eStage I \u0026amp; II\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003eStage III \u0026amp; IV\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e*Mann-Whitney U Test \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e**Explore \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 5\u003c/strong\u003e Association Between \u003cem\u003eFOXF1\u003c/em\u003e gene Promoter Hypermethylation and Clinicopathological Characteristics\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable dir=\"rtl\" border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"636\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp; \u0026nbsp;P value*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"5\" valign=\"top\" style=\"width: 258px;\"\u003e\n \u003cp dir=\"LTR\"\u003ehypermethylation status**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" rowspan=\"2\" valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp dir=\"LTR\"\u003eNo. of patients\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" rowspan=\"2\" valign=\"top\" style=\"width: 174px;\"\u003e\n \u003cp dir=\"LTR\"\u003eCharacteristics\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 42px;\"\u003e\n \u003cp dir=\"LTR\"\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp; \u0026nbsp; Negative(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp dir=\"LTR\"\u003ePositive (%) \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp dir=\"LTR\"\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e0.61\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e0.14\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e0.53\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;0.74\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e0.34\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; 0.15\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp; \u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp; \u0026nbsp;\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e4(25%)\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e9(26.5%)\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e6(20%)\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e7(35%)\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e2(20%) \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;11(27.5%)\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp; 6(28.6%)\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e7(24.1%)\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e8(32%)\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e5(20%)\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;5(17.9%)\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e3(16.7%)\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; -\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; -\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e12(75%)\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e25(73.5%)\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e24(80%)\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e13(65%)\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e8(80%)\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e29(72.5%)\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; 15(71.4%)\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e22(75.9%)\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e17(68%)\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e20(80%)\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e23(82.1%) \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e19(83.3%)\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e0.06 (95%CI 0.01-13.1)\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;0.01 (95%CI: 0.0-0.02)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e16\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e34\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e30\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e20\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e27\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e23\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e21\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e29\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e25\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e25\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e28\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp dir=\"LTR\"\u003e\u003cstrong\u003eAge\u003c/strong\u003e\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026le; 50 years\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u0026gt;50 years\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u003cstrong\u003eGender\u003c/strong\u003e\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;Female\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;Male\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u003cstrong\u003eHistological\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Grade\u003c/strong\u003e\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;Poorly+Moderately\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;Well\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u003cstrong\u003eTumor location\u003c/strong\u003e\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003eDistal\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003eProximal\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u003cstrong\u003eTumor size\u003c/strong\u003e\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u0026le;10mm\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u0026gt;10mm\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u003cstrong\u003eTumor Stage\u003c/strong\u003e\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;Stage I \u0026amp; II\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;Stage III \u0026amp; IV\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u003cstrong\u003eMethylation Levels\u003c/strong\u003e\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;(PMR Values)\u003c/strong\u003e\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003eMedian PMR (CRC Patients) \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003eMedian PMR (Healthy Controls)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;* \u0026nbsp;Fisherʼs \u003csup\u003e\u0026nbsp;\u003c/sup\u003eExact Test\u003c/p\u003e\n\u003cp\u003e**Crosstabs\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFOBT\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eresults\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe also examined the\u0026nbsp;FOBT\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eas an early diagnostic test. The sensitivity of FOBT was 38% compared to 59% for \u003cem\u003eFOXF1\u003c/em\u003e gene promoter methylation. These results showed that the sensitivity of \u003cem\u003eFOXF1\u003c/em\u003e gene promoter methylation in stool samples was significantly higher than that of the FOBT.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eColorectal cancer is a major public health concern, accounting for about 10% of worldwide incidence and mortalities (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). In the development of CRC, there is a gradual accumulation of different genetic and epigenetic changes, with abnormal DNA methylation being a crucial mechanism (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Aberrant DNA methylation patterns are commonly observed in gastrointestinal neoplasms and could serve as valuable markers for screening, diagnosis, prognosis determination, and prediction of CRC. In addition, the plentiful presence in extracellular environments, resistance to degradation, and presence in plasma, serum, feces specimens, and other noninvasive bodily fluids facilitates quantitative analysis of methylated DNA biomarkers (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Collecting bodily fluids is a non-intrusive process that allows for the early identification of cancer or disease monitoring (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Numerous studies have validated stool-based methylated DNA detection as an effective method for early CRC screening (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Promoter hypermethylation contributes to the inactivation of tumor suppressor genes and ultimately oncogenic processes by affecting key cellular pathways (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Common methylated and silenced genes often seen in CRC include vimentin(VIM), cadherin-1(CDH1), MLH1, TIMP metallopeptidase inhibitor-3 (TIMP3), hypermethylated in cancer-1 (HIC1) and secreted frizzled related protein-1(SFRP1) (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Recently, methylated syndecan-2 (mSDC2) and methylated SEPT9 (mSEPT9) have identified as promising biomarkers for non-invasive detection of CRC (\u003cspan additionalcitationids=\"CR35 CR36\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). Researchers have studied abnormal methylation of different genes in the tissues and bodily fluids of individuals with CRC, which could be used as valuable biomarkers in screening for CRC (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e).Cologuard is the first multitarget stool DNA test that has received FDA approval for CRC screening. It evaluates KRAS mutations, as well as BMP3 and NDRG4 methylation levels (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Moreover, NDRG4, BMP3, and SEPT9 are now incorporated as DNA methylation markers in FDA-approved CRC screening tests (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). Therefore, we aimed to identify non-invasive biomarkers capable of detecting CRC in small stool samples. Analyzing single genes in epigenetic studies could provide a more cost-effective option for screening. Nevertheless, these studies have presented remarkable initial findings about these single epigenetic markers and their utility as stool DNA\u0026ndash;based assays (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). Moradi et al. reported that PMR values in the stool of CRC patients was higher than those of healthy controls. Also, the analysis of SOX21 gene promoter methylation using a stool-based assay shows a notably high level of sensitivity (72.5%) and specificity (100%) (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). Zhang et al. showed that SPG20 promoter hypermethylation serves as a highly sensitive and specific biomarker in the detection of CRC using stool samples (80.2% and 100%, respectively) (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). Gl\u0026ouml;ckner et al. in stage I to III CRC patients, TFPI2 methylation was found in stool DNA with a sensitivity ranging from 76\u0026ndash;89% and a specificity ranging from 79\u0026ndash;93%. recognition of TFPI2 methylation in fecal DNA may serve as a biomarker for CRC screening (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e).Muller et al. identified a sensitivity of 77\u0026ndash;90% and a specificity of 77% for SFRP2 methylation as a sensitive single DNA-based biomarker for detecting CRC in stool samples from 49 patients (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). According to research single marker methylation-based stool using Vimentin and SFRP2, the sensitivity has found to be 52% and 87\u0026ndash;94%, while the specificity has reported 90% and 85\u0026ndash;90%, respectively (\u003cspan additionalcitationids=\"CR45 CR46\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). Various studies have indicated that the FOX family plays a crucial role in cellular proliferation, apoptosis, metastasis, the cell cycle, cellular metabolism, progression, and cancer biology in various cancers (\u003cspan additionalcitationids=\"CR49 CR50\" citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e). In total, Dysregulation of FOX proteins may impact the cell fate, tumor growth, and metastasis of cancer (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e). \u003cem\u003eFOXF1\u003c/em\u003e, a transcription factor, attaches to the promoter regions of target genes to control gene expression levels and influence the function of various signaling pathways crucial for physiological and pathological processes (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). \u003cem\u003eFOXF1\u003c/em\u003e is critical for controlling angiogenesis during embryonic development and may also be involved in angiogenesis, impacting CRC progression. Increased \u003cem\u003eFOXF1\u003c/em\u003e expression has been linked to aggressive CRC features, including poor differentiation and advanced disease stages. There was a significant association between increased \u003cem\u003eFOXF1\u003c/em\u003e expression and poorer prognosis in CRC patients (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). In contrast, \u003cem\u003eFOXF1\u003c/em\u003e expression is often silenced through DNA hypermethylation in breast cancer, where it acts as a tumor suppressor (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e). Herrera-Merchan et al. reported that \u003cem\u003eFOXF1\u003c/em\u003e expression could also be silenced via promoter methylation in lung adenocarcinoma and functions as a tumor suppressor (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e). In another study, Mitchell et al. analyzed a panel of 23 genes in the blood and tissue of CRC patients compared to a control group. They found increased DNA methylation in at least half of CRC tissue samples compared to normal tissue. Additionally, 11 genes, including \u003cem\u003eFOXF1\u003c/em\u003e, exhibited low methylation levels in peripheral blood DNA from healthy individuals (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e). On the other hand, \u003cem\u003eFOXF1\u003c/em\u003e promotes the development and maintenance of gastrointestinal stromal tumors through the regulation of KIT and ETV1 expression (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e). In hepatocellular carcinoma, \u003cem\u003eFOXF1\u003c/em\u003e serves as a suppressor of cell invasion and tumorigenesis, with its expression closely linked to patient survival rates (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e). Furthermore, in CRC cell lines with p53 inactivation, \u003cem\u003eFOXF1\u003c/em\u003e expression is silenced, leading to genomic instability in \u003cem\u003eFOXF1\u003c/em\u003e-expressing CRC cells with a dysfunctional p53-p21WAF1 checkpoint. This underscores \u003cem\u003eFOXF1'\u003c/em\u003es significance in CRC development (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e). Additionally, the competitive binding of miR-375-3p by LINC00022 could lead to the up-regulation of \u003cem\u003eFOXF1\u003c/em\u003e, potentially contributing to CRC progression. (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e). Lo et al. reported cytoplasmic mislocalization of upregulated \u003cem\u003eFOXF1\u003c/em\u003e protein in CRC epithelial cells, which is associated with poor prognosis factors (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Furthermore, \u003cem\u003eFOXF1\u003c/em\u003e upregulation is related to poorer diagnosis, the advanced progression, invasion, and metastasis of CRC (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Recently reported that \u003cem\u003eFOXF1\u003c/em\u003e gene methylation levels in the plasma of CRC patients are higher than healthy controls (\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e). In the present study, for the first time, we determined the \u003cem\u003eFOXF1\u003c/em\u003e gene promoter methylation for 50 stool samples patients with CRC and 50 healthy controls. Our results showed that The median PMR values of stool samples from patients and healthy controls were between 0.01 to 13.1 and 0.0 to 0.02, respectively. In addition, we examined the association of the PMR values obtained from stool samples with the demographic and pathologic features of the patients. There was no statistically significant correlation found between the methylation levels of the \u003cem\u003eFOXF1\u003c/em\u003e gene promoter and age, gender, histological grade, tumor stage, tumor location, or tumor size in CRC patients. In our research, \u003cem\u003eFOXF1\u003c/em\u003e gene promoter methylation position was assessed by the MethyLight technique, a quantitative method with high sensitivity and specificity capable of assaying a small amount of DNA. Our data revealed that the \u003cem\u003eFOXF1\u003c/em\u003e gene promoter methylation had higher sensitivity and specificity than FOBT in stool samples.\u003c/p\u003e \u003cp\u003eAlthough our results offer valuable information into the potential of FOXF1 methylation as a noninvasive biomarker for CRC detection in stool samples, certain limitations must be considered. the sample size is a relatively small and may affect the generalizability of our results. Furthermore, potential biases in patient selection and sample collection could impact the observed methylation patterns. To validate our findings and better evaluate the clinical utility of FOXF1 methylation in CRC detection, future studies should include larger, more diverse cohorts.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, as the first research to evaluate \u003cem\u003eFOXF1\u003c/em\u003e gene promoter methylation in stool samples of patients with CRC, it can be concluded that the PMR value for \u003cem\u003eFOXF1\u003c/em\u003e in CRC patients is higher than in healthy controls. The quantification of \u003cem\u003eFOXF1\u003c/em\u003e gene promoter methylation in stool samples shows promise as a non-invasive biomarker for CRC screening, with a sensitivity of 59% and a specificity of 96%. These results suggest potential sensitivity advantages over FOBT. Additionally, \u003cem\u003eFOXF1\u003c/em\u003e gene promoter methylation was found to be independent of age, sex, tumor grade, or stage, highlighting its potential utility in diverse patient populations. Future research with larger cohorts and more diverse populations is necessary to confirm these findings and explore its feasibility as a cost-effective screening tool.\u003c/p\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eLimitations\u003c/h2\u003e \u003cp\u003eThe study has several limitations that must be addressed to strengthen its findings. One key limitation is the relatively small number of participants in the study, which may have reduced its statistical power. Moreover, the limited sample size could impact the generalizability of the results to a wider population Therefore, it is crucial to conduct further research with a larger sample size to enhance the validity and practicality of our results. Another limitation is that several important risk factors for colorectal cancer, such as smoking, diet, and alcohol consumption, were not investigated due to insufficient clinical data. Furthermore, the research was carried out at a lone center. So The homogeneity of the population and inadequate analysis of confounding factors may also affect the reliability of the results. Furthermore, focusing solely on \u003cem\u003eFOXF1\u003c/em\u003e methylation without exploring additional biomarkers reduces the potential diagnostic accuracy. Methodological constraints, such as the standardization of stool sample collection, and the absence of a cost-effectiveness analysis further limit the study's real-world applicability. Future work should address these issues through larger, more diverse cohorts and comprehensive validation studies. Methylation-based evaluation is susceptible to false-positive and false-negative results due to various biological and technical factors. For example, contamination of stool samples with residual methylated DNA from food or external sources can lead to false positives. Also, Minimal amounts of DNA may be shed into the stool in the early stages of the disease, which reduces the ability to detect methylated DNA and can lead to false-negative.\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCRC: Colorectal cancer\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eFOXF1\u003c/em\u003e: \u0026nbsp;Forkhead box F1\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFOBT: \u0026nbsp; \u0026nbsp; Fecal occult blood test\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePMR: \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003ePercentage of the methylated reference\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCIMP: \u0026nbsp; \u0026nbsp; CpG Island Methylator Phenotype\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are grateful to the staff at the Medical Genetics Laboratory, Reference Laboratory, and Kermanshah University of Medical Sciences for their support throughout this research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBayan Mehrjoei󠅥 and Nayebali Rezvani designed the research.\u003c/p\u003e\n\u003cp\u003eSusan Tahmasebi\u0026nbsp;performed stool samples and clinical data collection; Nayebali Rezvani analyzed data; Bayan Mehrjoei󠅥 wrote the paper.\u0026nbsp;Asad Vaisi-Raygani\u0026nbsp;edited the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSource of Funding:\u0026nbsp;\u003c/strong\u003eNo funding\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest:\u003c/strong\u003eThere are no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll individuals partaking were tasked with signing the informed consent form.\u0026nbsp;The study was approved by the Ethics Committee at Kermanshah University of Medical Sciences Iran (Ethics code: no 95285) and conformed to the principles of the Helsinki Declaration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Data is provided within the manuscript or supplementary information files\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMehrjoei B, Haghnazari L, Bashiri H, Rezvani N. 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BMC Cancer. 2022;22(1):453.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDastafkan Z, Rezvani N, Amini S. Diagnostic value of FOXF1 gene promoter-methylated DNA in the plasma samples of patients with colorectal cancer. Int J Biol Mark. 2023;38(3\u0026ndash;4):194\u0026ndash;202.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Colorectal cancer, Epigenetic, Biomarkers, DNA methylation, FOXF1 gene","lastPublishedDoi":"10.21203/rs.3.rs-5551296/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5551296/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eColorectal cancer (CRC) pathogenesis is influenced by genes promoter region methylation, a key epigenetic mechanism. We sought to determine whether \u003cem\u003eFOXF1\u003c/em\u003epromoter methylation in stool samples could serve as a CRC biomarker.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterials and methods:\u003c/strong\u003e We analyzed \u003cem\u003eFOXF1\u003c/em\u003e gene promoter methylation in stool samples from 50 CRC patients and 50 healthy controls. The percentage of methylation reference (PMR) value was determined for each sample.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e PMR levels of the \u003cem\u003eFOXF1\u003c/em\u003e gene were higher in CRC patients than in controls (P\u0026lt;0.001). Median PMR values were 0.06 (95% CI 0.01-13.1) in stool samples from CRC patients and 0.01 (95% CI 0.0-0.02) in controls. Receiver Operating Characteristics (ROC) Curve analysis displayed a sensitivity of 59% and specificity 96% for \u003cem\u003eFOXF1\u003c/em\u003e gene methylation in stool samples.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e The elevated \u003cem\u003eFOXF1\u003c/em\u003e methylation levels in CRC stool samples, along with the test's sensitivity and specificity, suggest that this gene may be utilized as a non-invasive biomarker for colorectal cancer detection, independent of sex, age, or disease stage.\u003c/p\u003e","manuscriptTitle":"Evaluation of FOXF1 promoter DNA methylation, as a promising epigenetic biomarker for colorectal cancer in stool samples","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-21 15:25:07","doi":"10.21203/rs.3.rs-5551296/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"cf0a24a0-ec62-4ff8-af7a-b8ab8cb963f5","owner":[],"postedDate":"March 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-04-04T11:39:27+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-21 15:25:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5551296","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5551296","identity":"rs-5551296","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}