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: Name of Journal: World Journal of Gastroenterology
Manuscript NO: 46439
ManuscriptType: ORIGINAL ARTICLE
Retrospective Study
Clinical value of preoperative methylated septin 9 in Chinese colorectal cancer patients
Xue Y et al. Prognostic biomarker of colorectal cancer
Xue Yang, Zhi-Jie Xu, Xi Chen, Shuang-Shuang Zeng, Long Qian, Jie Wei, Mei Peng, Xiang Wang, Wan-Li Liu, Hong-Ying Ma, Zhi-Cheng Gong, Yuan-Liang Yan
Xue Yang, Xi Chen, Shuang-Shuang Zeng, Long Qian, Jie Wei, Mei Peng, Xiang Wang, Wan-Li Liu, Hong-Ying Ma, Zhi-Cheng Gong, Yuan-Liang Yan, Department of Pharmacy, Institute for Rational and Safe Medication Practices, National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Central South University, Changsha 410008, Hunan Province, China
Zhi-Jie Xu, Department of Pathology, Xiangya Hospital, Central South University, Changsha 410008, Hunan Province, China
ORCID number: Xue Yang (0000-0002-7856-5824); Zhi-Jie Xu ( HYPERLINK "http://orcid.org/0000-0003-2047-883X" \t "https://www.f6publishing.com/Forms/Manuscript/Author/_blank" 0000-0003-2047-883X); Xi Chen (0000-0001-6702-2771); Shuang-Shuang Zeng (0000-0003-3152-0589); Long Qian ( HYPERLINK "http://orcid.org/0000-0002-1819-5411" \t "https://www.f6publishing.com/Forms/Manuscript/Author/_blank" 0000-0002-1819-5411); Jie Wei (0000-0002-5554-0405); Mei Peng (0000-0001-6381-4958); Xiang Wang (0000-0001-6222-8033); Wan-Li Liu (0000-0002-7315-7856); Hong-Ying Ma (0000-0002-0589-3456); Zhi-Cheng Gong ( HYPERLINK "http://orcid.org/0000-0002-2404-0942" \t "https://www.f6publishing.com/Forms/Manuscript/Author/_blank" 0000-0002-2404-0942); Yuan-Liang Yan (0000-0001-6610-3617).
Author contributions: Yang X and Xu ZJ designed the research scheme and contributed equally to this article; Yan YL and Gong ZC contributed equally to correspondence about this manuscript; Chen X, Zeng SS, Qian L, and Wei J collected data from the samples; Peng M, Wang X, Liu WL, Xu ZJ, and Ma HY analyzed the data.
Supported by the National Natural Science Foundation of China, No. 81803035, No. 81703036, and No. 81572946; the China Postdoctoral Science Foundation, No. 2017M610510; and the Youth Fund of Xiangya Hospital, No. 2017Q17.
Institutional review board statement: This study was reviewed and approved by the Ethical Committee of Xiangya Hospital of Central South University (Approval No. 2018111100).
Informed consent statement: According to the Human Biomedical Research Ethical Review Procedures approved by the National Health and Family Planning Committee of China (No. 11, Section 39), informed consent was waived because of the retrospective nature of the study. After the following circumstances have been reviewed and approved by the ethics committee, the informed consent form can be waived if: Research is conducted using human body materials or data that can identify information, and the subjects cant be found, and the research project does not involve personal privacy and commercial interests.
Conflict-of-interest statement: The authors have no conflicts of interest to disclose.
Open-Access: This is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: HYPERLINK "http://creativecommons.org/licenses/by-nc/4.0/" http://creativecommons.org/licenses/by-nc/4.0/
Manuscript source: Unsolicited manuscript
Corresponding author: Zhi-Cheng Gong, PhD, Professor, Department of Pharmacy, Institute for Rational and Safe Medication Practices, National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Central South University, 87 Xiangya Road, Changsha 410008, Hunan Province, China. gongzhicheng@csu.edu.cn
Telephone: +86-731-84327306
Received: February 14, 2019
Peer-review started: February 15, 2019
First decision: March 5, 2019
Revised: March 25, 2019
Accepted: April 10, 2019
Article in press:
Published online:
Abstract
BACKGROUND
The methylated septin 9 (mSEPT9) assay was the first blood-based test approved by the United States Food and Drug Administration as a colorectal screening test. However, the diagnostic and prognostic role of preoperative mSEPT9 for colorectal cancer (CRC) in Chinese patients is still unknown.
AIM
To improve the understanding of diagnostic and prognostic factors, serum mSEPT9 was detected in Chinese CRC patients.
METHODS
A retrospective analysis of 354 cases, of which 300 had CRC and 54 were normal, was performed in China. Patients characteristics, treatments, and laboratory data, including age, the date of surgery, Union for International Cancer Control (UICC) stages, distant metastasis (M), and so on, were collected. Methylation levels of SEPT9 were quantified by quantitative, methylation-specific polymerase chain reaction before surgery. In addition, the effects of mSEPT9 on the occurrence and prognosis of 330 CRC cases from The Cancer Genome Atlas (TCGA) database were evaluated using bioinformatics analyses. Potential prognostic factors for overall survival (OS) and progression-free survival (PFS) were evaluated by Kaplan-Meier univariate analysis.
RESULTS
In Chinese CRC patients, positive mSEPT9 was strongly associated with advanced UICC stages, deeper invasion by the primary tumor, and more distant metastasis. Methylation levels of SEPT9 were stage-dependent and showed a stepwise increase in UICC stages (IIV), primary tumor categories (T1T4), regional node categories (N0N2), and distant metastasis categories (M0M1). The patients with positive mSEPT9 showed a tendency toward lower PFS. After analyzing TCGA clinical data, the high mSEPT9 group was found to be obviously correlated only with more distant metastasis. The patients with high mSEPT9 levels showed a tendency toward lower OS. Besides, nine meaningful mSEPT9 sites were found to provide guidance for the follow-up studies.
CONCLUSION
MSEPT9 analysis may add valuable information to current tumor staging. Serum mSEPT9 in Chinese CRC patients appears to offer promising novel prognostic markers and might be considered for monitoring CRC recurrence.
Key words: Methylated septin 9; Methylated; Colorectal cancer; Diagnosis; Prognosis
The Author(s) 2019. Published by Baishideng Publishing Group Inc. All rights reserved.
Core tip: This study retrospectively explored the value of serum septin 9 methylation (mSEPT9) in the diagnosis and prognosis of colorectal cancer in a Chinese population. Preoperative mSEPT9 levels in 354 enrolled patients were retrospectively analyzed. In addition, the effects of mSEPT9 on the occurrence and prognosis of 330 colorectal cancer cases from The Cancer Genome Atlas database were evaluated using bioinformatics analyses. Besides, nine meaningful mSEPT9 sites were found to provide guidance for the follow-up studies.
Yang X, Xu ZJ, Chen X, Zeng SS, Qian L, Wei J, Peng M, Wang X, Liu WL, Ma HY, Gong ZC, Yan YL. Clinical value of preoperative methylated septin 9 in Chinese colorectal cancer patients. World J Gastroenterol 2019; In press
INTRODUCTION
Colorectal cancer (CRC) remains the third most common cancer expected to occur in men and women ADDIN EN.CITE Siegel201852[1]525217Siegel, Rebecca L.Miller, Kimberly D.Dvm, Ahmedin JemalCancer statistics, 2018Ca A Cancer Journal for CliniciansCa A Cancer Journal for Clinicians116812018[1], accounting for approximately 10% of the global cancer burden. To date, more than 90% of patients with early CRC have survived five years after diagnosis ADDIN EN.CITE 53[2, 3]5353017<global-cancer-facts-and-figures-3rd-edition.pdf>Siegel201454545417Siegel, RebeccaDesantis, CarolVirgo, KatherineStein, KevinMariotto, AngelaSmith, TenbroeckCooper, DexterGansler, TedLerro, CatherineFedewa, StaceyCancer treatment and survivorship statistics, 2012Ca Cancer J ClinCa Cancer J Clin252-2716442014[2,3]. However, in the case of regional spread to lymph nodes or adjacent organs, the five-year relative survival rate decreases to 69%, and when there is distant metastasis, it drops sharply to approximately 10% ADDIN EN.CITE Siegel201454[3, 4]545417Siegel, RebeccaDesantis, CarolVirgo, KatherineStein, KevinMariotto, AngelaSmith, TenbroeckCooper, DexterGansler, TedLerro, CatherineFedewa, StaceyCancer treatment and survivorship statistics, 2012Ca Cancer J ClinCa Cancer J Clin252-2716442014Rizell201388888817Rizell, SaraBarrens, Marie LouiseAndlinsobocki, AnnaStecksnblicks, ChristinaKjellberg, Heidrun45,X/46,XX karyotype mitigates the aberrant craniofacial morphology in Turner syndromeEuropean Journal of OrthodonticsEuropean Journal of Orthodontics467-4743542013[3,4]. Despite significant recent achievements in the diagnosis and treatment of these patients, resulting in partial reductions in overall incidence and mortality, there is no effective diagnostic assay so far for tumor progression or recurrence monitoring, especially in vitro.
Detection of CRC recurrences or metastases in the early stage by constant monitoring may improve long-term outcomes through timely treatment. The American Joint Committee on Cancer (AJCC) Cancer Staging, seventh edition has accepted clinically useful carcinoembryonic antigen (CEA) serum tumor marker as a site-specific prognostic factor in CRC ADDIN EN.CITE Cuccurullo201155[5]555517Cuccurullo, VincenzoAJCC Cancer Staging Handbook: from the AJCC Cancer Staging Manual (7th edition)European Journal of Nuclear Medicine & Molecular ImagingEuropean Journal of Nuclear Medicine & Molecular Imaging408-4083822011[5]. However, the low detection sensitivity of CEA hinders its use for many surgical patients, because patients with negative CEA results before surgery usually cannot be monitored after surgery ADDIN EN.CITE Young201656[6, 7]565617Young, G. P.Pedersen, S. K.Mansfield, SMurray, D. H.Baker, R. T.Rabbitt, PByrne, SBambacas, LHollington, PSymonds, E. L.A cross-sectional study comparing a blood test for methylated BCAT1 and IKZF1 tumor-derived DNA with CEA for detection of recurrent colorectal cancerCancer MedCancer Med2763-27725102016Nicholson201557575717Nicholson, B. D.Shinkins, BPathiraja, IRoberts, N. W.James, T. J.Mallett, SPerera, RPrimrose, J. N.Mant, DBlood CEA levels for detecting recurrent colorectal cancerCochrane Database of Systematic ReviewsCochrane Database of Systematic ReviewsCD0111342015122015[6,7]. In addition, periodic computed tomography (CT) scanning is another noninvasive method for surgical therapeutic effect assessment ADDIN EN.CITE Ng201758[8]585817Ng, Sarah B.Chua, ClarindaNg, MatthewGan, AnnaPoon, Polly SyTeo, MelissaFu, CherylinWei, Qiang LeowLim, Kiat HonChung, AlexanderIndividualised multiplexed circulating tumour DNA assays for monitoring of tumour presence in patients after colorectal cancer surgeryScientific ReportsScientific Reports4073772017[8]. However, CT scans have limited sensitivity and high false positive rates, and cannot be used routinely as a monitoring examination due to the danger of long-term radiation ADDIN EN.CITE Metser201059[9]595917Metser, UYou, J.Mcsweeney, SFreeman, MHendler, AAssessment of tumor recurrence in patients with colorectal cancer and elevated carcinoembryonic antigen level: FDG PET/CT versus contrast-enhanced 64-MDCT of the chest and abdomenAjr American Journal of RoentgenologyAjr American Journal of Roentgenology766-7119432010[9]. Therefore, development of novel, sensitive biomarkers for monitoring recurrences or metastases of CRC is urgently needed.
Hypermethylation of the promoter of septin 9 (SEPT9) has previously been shown to be a sensitive and specific biomarker in various cancers including CRC[10-13] and its precursor lesions ADDIN EN.CITE Li201466[16]666617Li, Y.Song, L.Gong, Y.He, B.Detection of colorectal cancer by DNA methylation biomarker SEPT9: past, present and futureBiomarkers in MedicineBiomarkers in Medicine755-769852014[14-16]. As a result, the methylated SEPT9 (mSEPT9) assay became the first blood-based test approved by the United States Food and Drug Administration as a CRC screening test ADDIN EN.CITE Li201467[17]676717Li, YanwenLi, YuehuiLiu, YanhongXie, PingliLi, FengLi, GuanchengPAX6, a Novel Target of microRNA-7, Promotes Cellular Proliferation and Invasion in Human Colorectal Cancer CellsDigestive Diseases & SciencesDigestive Diseases & Sciences598-6065932014[17]. A study of Korean CRC patients found that serum mSEPT9 had a tendency to show metastasis and a low disease-free survival rate ADDIN EN.CITE Lee201368[18]686817Lee, Hye SeungSang, Mee HwangKim, Taek SooKim, Duck WooPark, Do JoongKang, Sung BumKim, Hyung HoPark, Kyoung UnCirculating Methylated Septin 9 Nucleic Acid in the Plasma of Patients with Gastrointestinal Cancer in the Stomach and ColonTranslational OncologyTranslational Oncology290-U245632013[18]. In a recent study of German CRC patients, mSEPT9 was significantly associated with Union for International Cancer Control (UICC) stages both before and after therapy ADDIN EN.CITE Bergheim201869[19]696917Bergheim, JSemaan, AGevensleben, HGroening, SKnoblich, ADietrich, JWeber, JKalff, J. C.Bootz, FKristiansen, GPotential of quantitative SEPT9 and SHOX2 methylation in plasmatic circulating cell-free DNA as auxiliary staging parameter in colorectal cancer: a prospective observational cohort studyBritish Journal of CancerBritish Journal of Cancer2018[19]. In addition, quantitative mSEPT9 levels have been successfully applied for the diagnosis of CRC ADDIN EN.CITE Wu201771[21]717117Wu, AnshanWu, AnshanHe, SiqiHe, SiqiLi, JingjingLi, JingjingLiu, LingLiu, LingLiu, ChunlanLiu, ChunlanColorectal cancer in cases of multiple primary cancers: Clinical features of 59 cases and point mutation analysesOncology LettersOncology Letters4720-47261362017[19-22], and for the screening, diagnosis, monitoring, prognosis, and molecular staging of head and neck squamous cell carcinomas (HNSCC) ADDIN EN.CITE Schrck201770[20]707017Schrck, ALeisse, ADe, Vos LGevensleben, HDrge, FFranzen, AWachendrfer, MSchrck, FEllinger, JTeschke, MFree-Circulating Methylated DNA in Blood for Diagnosis, Staging, Prognosis, and Monitoring of Head and Neck Squamous Cell Carcinoma Patients: An Observational Prospective Cohort StudyClinical ChemistryClinical Chemistry12886372017[19]. However, the diagnostic and prognostic role of preoperative mSEPT9 for CRC in Chinese patients is still unknown.
This study assessed the correlation between clinicopathological characteristics and preoperative serum mSEPT9 in Chinese CRC patients and, further, to confirm the correlation between mSEPT9 levels and CRC prognosis by bioinformatics analyses. In addition, we analyzed methylated sites that were co-upregulated or co-downregulated in colon and rectum tumors, to provide the theoretical guidance for further research.
MATERIALS AND METHODS
Patients and samples
This present study was conducted from December 2017 to November 2018 among patients at the Department of Hepatobiliary and Enteric Surgery in Xiangya Hospital. A total of 354 subjects with mSEPT9 serum detection before surgery were recruited from a medicine-pharmacy-nursing integrative parenteral medication rational use and safety early warning platform, the Parenteral Prescription Early Warning and Assessment System, including 300 CRC patients and 54 normal subjects. This study was approved by the Ethical Committee of Xiangya Hospital of Central South University (Approval No. 2018111100).
Three hundred patients presented with histologically confirmed primary CRC. Recurrences or metastases were determined from diagnostic tests (CT scan, magnetic resonance imaging, or colonoscopy) and confirmed through tissue pathology when available ADDIN EN.CITE Young201656[6]565617Young, G. P.Pedersen, S. K.Mansfield, SMurray, D. H.Baker, R. T.Rabbitt, PByrne, SBambacas, LHollington, PSymonds, E. L.A cross-sectional study comparing a blood test for methylated BCAT1 and IKZF1 tumor-derived DNA with CEA for detection of recurrent colorectal cancerCancer MedCancer Med2763-27725102016[7]. Clinical parameters, including mSEPT9 detection results, gender, age, UICC stage, histologic grade, primary tumor (T) categories, regional node (N) categories, distant metastasis categories (M), lymphatic invasion (L), lymph nodal status, vascular invasion (V), and tumor site, were collected. The UICC stage, tumor node metastasis (TNM) categories and histologic differentiation were graded on the basis of the eighth edition of the AJCC ADDIN EN.CITE Pollaers201872[22]727217Pollaers, KatherineHinton-Bayre, AntonFriedland, Peter L.Farah, Camile S.AJCC 8th Edition oral cavity squamous cell carcinoma staging Is it an improvement on the AJCC 7th Edition?Oral OncologyOral Oncology23-28822018[23]. Progression-free survival (PFS) time was calculated from the CRC patients date of surgery to presentation of clinical or pathological evidence of cancer recurrence.
In addition, 330 colorectal adenocarcinomas from The Cancer Genome Atlas (TCGA) Research Network (http://cancergenome.nih.gov/.) were selected and analyzed retrospectively. Patients whose mSEPT9 levels were less than or equal to median were assigned to the low mSEPT9 group, whereas others were assigned to the high mSEPT9 group. The overall survival (OS) time was calculated from the CRC patients date of surgery to the date of dead or to the last contact date.
Methylated SEPT9 detection
A 10 mL peripheral blood sample was collected with a 10 mL K2EDTA anticoagulant tube for the SEPT9 assay [BioChain (Beijing) Science and Technology, Inc., Beijing, China]. Peripheral blood sample storage and transportation, DNA extraction, and bisulfite conversion were performed manually following the manufacturers instructions of the Epi proColon 2.0 kit (Epigenomics AG, Berlin, Germany). The mSEPT9 was assayed with the Epi proColon 2.0 kit on an AB7500 Fast Dx Real Time polymerase chain reaction device (Life Technologies) in the Clinical Laboratory of Xiangya Hosp i t a l , C e n t r a l S o u t h U n i v e r s i t y . B r i e f l y , a p o l y m e r a s e c h a i n r e a c t i o n ( P C R ) t e s t w a s p e r f o r m e d i n t r i p l i c a t e w i t h 1 5 L t e m p l a t e D N A p e r w e l l a n d r u n f o r 4 5 c y c l e s [ 2 4 ] . T h e i n s t r u m e n t s o f t w a r e w a s u s e d t o r e c o r d t h e P C R r e s u l t s f o r - a c t i n ( A C T B ) a n d m e t h y lated SEPT9 from each of the triplicate reactions. The validity of each sample batch was determined according to methylated SEPT9 and ACTB threshold count (Ct) values for the positive and negative controls. ACTB served as an internal reference to assess the integrity of each sample. According to the instructions, Ct value was less than 41.1 was assigned to the positive mSEPT9 group, whereas those whose Ct value was over 41.1 were assigned to the negative mSEPT9 group.
Statistical analyses
All statistical analyses were performed using SPSS 18 software (SPSS Inc, Chicago, United States). The measurable data was expressed as the mean and standard deviation (SD). Differences of clinicopathological characteristics and Ct values between groups were compared via t-tests and 2 t e s t . T h e u n i v a r i a t e a n a l y s i s w a s p e r f o r m e d t o a s s e s s t h e e f f e c t o f m S E P T 9 t o p r e d i c t P F S a n d O S b y t h e K a p l a n - M e i e r m e t h o d . B i n a r y l o g i s t i c r e g r e s s i o n w a s u s e d t o a n a l y z e t h e a s s o c i a t i o n b e t w e e n e a c h g e n e t i c b i o m a r k e r ( e . g . , m i s m a t c h e d r e p a i r p r o t e i n s ) and mSEPT9. All the statistical tests were bilateral, and P < 0.05 was considered statistically significant.
RESULTS
The mSEPT9 in CRC patients and normal subjects
Among Chinese CRC patients, the methylated Ct values of 300 primary CRC patients and 54 normal subjects were analyzed. Based on contradictory trends for Ct value and expression, the preoperative serum mSEPT9 levels were significantly higher in CRC patients than in the normal subjects (P = 0.008) (Figure 1A). The positive rate of mSEPT9 was 52.3% for CRC patients and 25.9% for normal subjects (P = 0.102) (Figure 1B).
Among 351 patients from the TCGA database, the mSEPT9 levels of 330 CRC patients and 21 normal subjects were analyzed. The serum mSEPT9 levels of the CRC patients were higher than those of the normal subjects, but were not statistically significant (P = 0.530) (Supplemental Figure 1).
Clinicopathological characteristics and mSEPT9 in CRC
The Chinese CRC patients, clinicopathological features of 300 CRC patients are described in Table 1. As shown in Figure 1C, patients older than 50 years were statistically more numerous than those aged 50 or younger in both positive and negative groups (P = 0.016). Through analyzing UICC stages, we found that the positive rate of stage III was observably higher than stage I (46.6% vs 31.0%, P = 0.012) (Figure 2A); mSEPT9 levels showed a significant increase from UICC stages II to III (P = 0.033) and stages III to IV (P < 0.0001), but no obvious difference was detected between stages I to II (P = 0.898, Figure 3A).
In addition, the association of mSEPT9 levels and rate of positive mSEPT9 among primary tumor categories (T1T4), regional node categories (N0N2) and distant metastasis categories (M0M1) were also analyzed. The detection rate of positive T3 was observably higher than that of T1 (51.1% vs 40.0%, P = 0.019) (Figure 2B). Positive rate and levels of mSEPT9 revealed a significant increase from T3 to T4 (P = 0.030, P = 0.046, respectively) (Figure 2B, Figure 3B). In terms of regional node categories, N0 to N2 showed a gradual increase in mSEPT9 levels (P = 0.012) (Figure 3C), but did not show any association with the rate of positive mSEPT9 (Figure 2C). As shown in Figures 2D and 3D, mSEPT9 showed the best ability to discriminate between local and metastatic CRC (P = 0.015, P < 0.0001, respectively). However, higher mSEPT9 levels were not found in CRC patients with lymphatic or vascular invasion than in those without invasion (all P > 0.05). We also failed to find association among MLH1, MSH2 (25D12), MSH6, PMS2, and Ki67 and mSEPT9 (all P > 0.05) (Supplemental Table 1).
The clinicopathological features of 330 CRC patients from the TCGA database are described in detail in Supplemental Table 2. Similarly, there was a tendency for more distant metastasis (P = 0.0001) and more CRC patients older than 50 (P < 0.0001) in the high mSEPT9 group, but no significant difference was found in UICC stages, primary tumor categories, or regional node categories (all P > 0.05).
Prognostic significance of mSEPT9 in CRC patients
Kaplan-Meier univariate analysis showed that positive mSEPT9 was obviously associated with shorter PFS among the Chinese CRC patients (P = 0.019, Figure 4A). The positive mSEPT9 CRC cases were estimated to have an mean PFS duration of 3.7 mo [95% confidence interval (CI): 2.14-5.19] compared with the 6.0 mo (95%CI: 0-13.87) in the negative mSEPT9 CRC cases.
In addition, serum mSEPT9 showed prognostic significance for the CRC patients from the TCGA database (P = 0.008, Figure 4B). CRC patients with low mSEPT9 levels were found to be correlated with longer OS. The low mSEPT9 CRC cases had an estimated mean OS duration of 8.1 months (95%CI: 6.53-9.27) compared with the 5.1 mo (95%CI: 3.87-6.33) in the high mSEPT9 CRC cases.
Significant methylation sites for SEPT9
In further analyzed TCGA clinical data, 124 mSEPT9 sites were found that showed HYPERLINK "http://www.baidu.com/link?url=YftxkOJHXGwrwMpLVEsDkuCsLMdvWRLxfkK4qUopRJ0QxyZSau_GwFuqKzSfTtd9VH6mGY9a7Bml5HIGa5iiKSd8PutvBqji_X3T7cdlYqPej8PtAYrqnx1q38ALV85q" \t "https://www.baidu.com/_blank" differential expression among normal subjects and those with colon and rectum adenocarcinoma, respectively (all P < 0.05) (Supplemental Figure 2). After analyzing the detailed information of these 124 mSEPT9 sites, 68 co-upregulated and 36 co-downregulated mSEPT9 sites in CRC adenocarcinoma were further observed. We finally confirmed that there were eight co-upregulated mSEPT9 sites (Figure 5) and one co-downregulated mSEPT9 site (cg02975107) through setting a cut-off of a two-fold expression change of mSEPT9.
DISCUSSION
Most patients with early CRC undergo curatively intended surgery to clear up primary lesions and local lymph node metastasis up. However, 30%-50% of patients would still confront tumor recurrence and might die from metastasis ADDIN EN.CITE Claudia201374[24]747417Claudia, AllemaniBernard, RachetWeir, Hannah KRichardson, Lisa CCme, LepageJean, FaivreGemma, GattaRiccardo, CapocacciaMilena, SantPaolo, BailiColorectal cancer survival in the USA and Europe: a CONCORD high-resolution studyInternational Journal of Cancer Journal International Du CancerInternational Journal of Cancer Journal International Du Cancer1170392013[25]. Timely monitoring of recurrence and metastasis is of great significance to the prognosis and survival of patients. In our study, mSEPT9 was proved to be an effective bio-marker for diagnosis, recurrence, and prognosis of CRC in Chinese patients, and nine significant mSEPT9 sites were confirmed for further in-depthconsideration.
Our study confirmed the value of serum mSEPT9 for CRC diagnosis. Compared with normal tissues in Chinese and TCGA data, serum SEPT9 was found to be hypermethylated in tumor tissues, which was consistent with previous studies[18,19,26]. Studies showed that age affected the detection rate of the SEPT9 assay[27,28], and we found that a positive rate of mSEPT9 was strongly associated with CRC patients aged over 50 years both in Chinese and TCGA data. This accords with the definition of an average risk population in National Comprehensive Cancer Network Guidelines for CRC[29]. Remarkably, we reported that SEPT9 performs outstandingly as an auxiliary molecular staging parameter in the Chinese population, especially because mSEPT9 levels could distinguish between pathological UICC and TNM stages in an incremental fashion. In addition, our data demonstrated that CRC patients in earlier tumor stages showed lower mSEPT9 levels compared to those with more advanced lesions, which is consistent with studies in German CRC patients ADDIN EN.CITE Chen201478[28]787817Chen, JianmeiWang, WeiningZhang, YangdeHu, TiehuiChen, YuxiangThe roles of miR-200c in colon cancer and associated molecular mechanismsTumour Biology the Journal of the International Society for Oncodevelopmental Biology & MedicineTumour Biology the Journal of the International Society for Oncodevelopmental Biology & Medicine64753572014[19,30-32]. Most importantly, its ability to identify patients with distant metastases emphasizes the potential of mSEPT9 as a bio-marker, which adds valuable information to the classification of tumors ADDIN EN.CITE Choi201579[29, 30]797917Choi, Audrey HNelson, Rebecca ASchoellhammer, Hans FCho, WonKo, MichelleArrington, AmandaOxner, Christopher R<