粪便中SFRP2基因甲基化改变作为大肠癌筛选标志物的研究
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摘要
目的和意义:
大肠癌是指大肠粘膜上皮在环境或遗传等多种致癌因素作用下发生的恶性病变,是世界上第三位最常见的肿瘤。结肠上皮细胞从异常增生经腺瘤阶段发展到癌变平均需10年时间,从可辨认的腺瘤发展为侵袭性癌需5年,这为早期筛查及预防提供了充分时间。在早期阶段大肠癌是最容易治疗的,但大肠癌起病隐匿,早期常无明显的临床表现,大约只有40%的患者在肿瘤局限期被检测出来,待出现症状大多已是中晚期。如能发现癌前病变(如腺瘤),通过及时的内镜下电凝切除即可阻止癌变的发生,大肠癌在早期临床阶段(如Ⅰ和Ⅱ期)被发现,肿瘤尚局限在肠壁中,外科手术治愈率分别为90%和75%,而进展型大肠癌预后则较差,有远处转移的患者五年生存率只有5%。通过检测和清除可能发展成为大肠癌的病变可以降低大肠癌的发病率及提高初级预防,并且早期发现大肠癌将会增加存活率。因此,对高危人群的早期筛查是防治大肠癌的关键。通过改变大规模人群的生活方式来减少大肠癌的发生率是难以实现的,而筛查可使大肠癌死亡率适度下降,并通过检测和去除大肠腺瘤(较少侵袭性的手术早期处理或内镜切除)而有可能降低大肠癌的发生率。现在大多数早期检测技术如大便潜血试验、气钡灌肠及电子纤维肠镜分别存在精确性低、费用高、侵袭性大、病人依从性不强甚至发生肠出血穿孔严重并发症等缺点,因而这些方法还不适合于筛查。因此,探索一种安全可靠、特异性强、能早期有效筛查大肠癌的方法成为医学界噬待解决的问题。肿瘤抑制基因CpG岛的超甲基化是大肠肿瘤发生的最常见的分子改变,许多基因的超甲基化与大肠癌的发生发展密切相关。由于甲基化改变往往发生在肿瘤变化的早期,易于在患者的粪便中检测到。DNA甲基化状态改变作为肿瘤标志物用于临床诊断,其特点和操作优于突变的检测。因而,DNA甲基化检测已成为探讨早期筛查大肠癌新的研究热点,它与大肠癌的早期诊断、分期、治疗及预后密切相关,甚至可用于大批人群的筛选。近年来,国内外学者发现大肠癌组织中分泌型卷曲相关蛋白2(SFRP2)基因启动子发生频繁的超甲基化而导致基因沉默,是大肠癌发生过程中的一个早期频繁事件。本研究通过检测大肠癌和大肠良性疾病患者粪便中SFRP2基因的甲基化状况,探讨粪便中SFRP2基因超甲基化作为大肠癌筛选标志物的可行性。
研究方法:
甲基化特异性PCR (MSP)技术分析69例大肠癌、34例腺瘤和26例增生性息肉患者的瘤组织和粪便中SFRP2基因启动子甲基化状态,30例内镜下正常的健康者作为对照。其中大肠癌患者的粪便分别于术前和术后第9日收集。同时,我们分析了SFRP2基因甲基化与肿瘤临床病理特点之间的关系。
研究结果:
SFRP2基因在91.3%(63/69)的大肠癌组织、79.4%(27/34)的腺瘤组织和53.8%(14/26)的增生性息肉组织中发生超甲基化,并在同一患者所对应的粪便中有87.0%(60/69)、61.8%(21/34)和42.3%(11/26)发生超甲基化。在正常对照的结肠粘膜组织中没有检测到SFRP2基因甲基化,但在粪便中有2例发生了SFRP2基因超甲基化。在大肠癌患者术前和术后第9天的粪便中SFRP2基因超甲基化有显著差异(P<0.001)。此外,SFRP2超甲基化与肿瘤的临床特征包括性别、年龄、肿瘤分期、位置及病理分级等无显著相关性。
结论:
粪便中SFRP2基因超甲基化是大肠癌的一种新的分子标记物,对于非侵袭性检测大肠癌及大肠良性病变具有高度的潜力。
Objective:
Colorectal cancer(CRC)is a malignant lesion occurres when the intestinal epithelium suffer from carcinogenic agents such as environmental or genetic factors,and is the third most common cancer worldwide. It takes ten years average that colonic epithelial cells developed from the adenomatous dysplasia neoplasia to carcinoma, and the identifiable adenoma development into invasive carcinoma needs five years, which provided sufficient time for early screening and prevention. CRC is most effectively treated when diagnosed at an early stage, but the early stage of CRC is mostly symptomless as well as delitescence nature. Approximately 40% of CRCs are diagnosed with localized disease, which have approximately a 70%~90% five-year survival rate after curative surgery. However, the prognosis worsens with advancing stage, and only 5% of patients diagnosed with distant metastases survive 5 years. With the potential of both reducing incidence from CRC as well as enhancing primary prevention through detection and removal of lesions that could potentially develop into cancer, early detection of CRC will increase survival the most. Therefore, the need for early detection is clear for high-risk groups, and an effective screening test would have substantial clinical benefits. It is difficult to reduce CRC incidence rate by changing people's way of life. Whereas CRC screening will moderate decline in mortality, and it may decrease the incidence of CRC through detection and removal of colorectal adenoma (early less invasive surgical treatment or endoscopic resection). Now that most of the early detection technologies such as fecal occult blood test, barium enema and electronic colonoscopy were low accuracy, high cost, invasive, non-compliance of patients, even serious complications such as intestinal bleeding and perforation and et al. So these methods is not suitable for screening. Therefore, exploration of a safe, reliable, specific, effective early CRC screening methods is the Urgent medical profession problem to be solved. CpG island hypermethylation of the tumor suppressor gene is the most common molecule change of CRC, and hypermethylation of the several genes is closely related to occurrence and development of CRC. Methylation changes often occurred in the early stage of CRC, which is easily detected in the faeces of patient. The characteristic and procedure of DNA methylation changes as tumor markers for the clinical diagnosis is superior to mutation detection. Thus, DNA methylation is closely correlated with early diagnosis, staging, treatment and prognosis of CRC and even can be used for large crowds screening, which has become a new research hot spots for early detection of CRC. In recent years, several studies have identified frequent promoter hypermethylation and silencing of the secreted frizzled-related protein 2 (SFRP2) genes in CRC, which is an early frequently event in colorectal tumorigenesis. This study is to investigate the feasibility of detecting hypermethylated SFRP2 gene in fecal DNA as a non-invasive screening tool for CRC by detecting faecal SFRP2 gene methylation status in patients with colorectal carcinoma and benign disease.
METHODS:
Methylation-specific PCR assay (MSP) was performed to analyze SFRP2 gene promoter methylation status in a blinded fashion in tumor tissues and in stool samples taken from 69 CRC patients preoperatively and at the 9th postoperative day, 34 patients with adenoma≥1 cm, 26 with hyperplastic polyp, and 30 endoscopically normal subjects. Simultaneously the relationship between hypermethylation of SFRP2 gene and clinicopathological features was analyzed.
RESULTS:
SFRP2 gene was hypermethylated in 91.3% (63/69) CRC, 79.4% (27/34) and 53.8% (14/26) adenoma and hyperplastic polyp tissues, and in 87.0% (60/69), 61.8% (21/34) and 42.3% (11/26) of corresponding fecal samples, respectively. In contrast, no methylated SFRP2 gene was detected in mucosal tissues of normal controls, while two cases of matched fecal samples from normal controls were detected with hypermethylated SFRP2. A significant decrease (P < 0.001) in the rate of hypermethylated SFRP2 gene was detected in the postoperative (8.7%, 6/69) fecal samples as compared with the preoperative fecal samples (87%, 60/69) of CRC patients. Moreover, no significant associations were observed between SFRP2 hypermethylation and clinicopathological features including sex, age, tumor stage, site, lymph node status and histological grade, etc.
CONCLUSION:
Hypermethylation of SFRP2 gene in fecal DNA is a novel molecular biomarker of CRC and carries a high potential for the remote detection of CRC and premalignant lesions as noninvasive screening method.
大肠癌是指大肠粘膜上皮在环境或遗传等多种致癌因素作用下发生的恶性病变,是世界上第三位最常见的肿瘤。结肠上皮细胞从异常增生经腺瘤阶段发展到癌变平均需10年时间,从可辨认的腺瘤发展为侵袭性癌需5年,这为早期筛查及预防提供了充分时间。在早期阶段大肠癌是最容易治疗的,但大肠癌起病隐匿,早期常无明显的临床表现,大约只有40%的患者在肿瘤局限期被检测出来,待出现症状大多已是中晚期。如能发现癌前病变(如腺瘤),通过及时的内镜下电凝切除即可阻止癌变的发生,大肠癌在早期临床阶段(如Ⅰ和Ⅱ期)被发现,肿瘤尚局限在肠壁中,外科手术治愈率分别为90%和75%,而进展型大肠癌预后则较差,有远处转移的患者五年生存率只有5%。通过检测和清除可能发展成为大肠癌的病变可以降低大肠癌的发病率及提高初级预防,并且早期发现大肠癌将会增加存活率。因此,对高危人群的早期筛查是防治大肠癌的关键。通过改变大规模人群的生活方式来减少大肠癌的发生率是难以实现的,而筛查可使大肠癌死亡率适度下降,并通过检测和去除大肠腺瘤(较少侵袭性的手术早期处理或内镜切除)而有可能降低大肠癌的发生率。现在大多数早期检测技术如大便潜血试验、气钡灌肠及电子纤维肠镜分别存在精确性低、费用高、侵袭性大、病人依从性不强甚至发生肠出血穿孔严重并发症等缺点,因而这些方法还不适合于筛查。因此,探索一种安全可靠、特异性强、能早期有效筛查大肠癌的方法成为医学界噬待解决的问题。肿瘤抑制基因CpG岛的超甲基化是大肠肿瘤发生的最常见的分子改变,许多基因的超甲基化与大肠癌的发生发展密切相关。由于甲基化改变往往发生在肿瘤变化的早期,易于在患者的粪便中检测到。DNA甲基化状态改变作为肿瘤标志物用于临床诊断,其特点和操作优于突变的检测。因而,DNA甲基化检测已成为探讨早期筛查大肠癌新的研究热点,它与大肠癌的早期诊断、分期、治疗及预后密切相关,甚至可用于大批人群的筛选。近年来,国内外学者发现大肠癌组织中分泌型卷曲相关蛋白2(SFRP2)基因启动子发生频繁的超甲基化而导致基因沉默,是大肠癌发生过程中的一个早期频繁事件。本研究通过检测大肠癌和大肠良性疾病患者粪便中SFRP2基因的甲基化状况,探讨粪便中SFRP2基因超甲基化作为大肠癌筛选标志物的可行性。
研究方法:
甲基化特异性PCR (MSP)技术分析69例大肠癌、34例腺瘤和26例增生性息肉患者的瘤组织和粪便中SFRP2基因启动子甲基化状态,30例内镜下正常的健康者作为对照。其中大肠癌患者的粪便分别于术前和术后第9日收集。同时,我们分析了SFRP2基因甲基化与肿瘤临床病理特点之间的关系。
研究结果:
SFRP2基因在91.3%(63/69)的大肠癌组织、79.4%(27/34)的腺瘤组织和53.8%(14/26)的增生性息肉组织中发生超甲基化,并在同一患者所对应的粪便中有87.0%(60/69)、61.8%(21/34)和42.3%(11/26)发生超甲基化。在正常对照的结肠粘膜组织中没有检测到SFRP2基因甲基化,但在粪便中有2例发生了SFRP2基因超甲基化。在大肠癌患者术前和术后第9天的粪便中SFRP2基因超甲基化有显著差异(P<0.001)。此外,SFRP2超甲基化与肿瘤的临床特征包括性别、年龄、肿瘤分期、位置及病理分级等无显著相关性。
结论:
粪便中SFRP2基因超甲基化是大肠癌的一种新的分子标记物,对于非侵袭性检测大肠癌及大肠良性病变具有高度的潜力。
Objective:
Colorectal cancer(CRC)is a malignant lesion occurres when the intestinal epithelium suffer from carcinogenic agents such as environmental or genetic factors,and is the third most common cancer worldwide. It takes ten years average that colonic epithelial cells developed from the adenomatous dysplasia neoplasia to carcinoma, and the identifiable adenoma development into invasive carcinoma needs five years, which provided sufficient time for early screening and prevention. CRC is most effectively treated when diagnosed at an early stage, but the early stage of CRC is mostly symptomless as well as delitescence nature. Approximately 40% of CRCs are diagnosed with localized disease, which have approximately a 70%~90% five-year survival rate after curative surgery. However, the prognosis worsens with advancing stage, and only 5% of patients diagnosed with distant metastases survive 5 years. With the potential of both reducing incidence from CRC as well as enhancing primary prevention through detection and removal of lesions that could potentially develop into cancer, early detection of CRC will increase survival the most. Therefore, the need for early detection is clear for high-risk groups, and an effective screening test would have substantial clinical benefits. It is difficult to reduce CRC incidence rate by changing people's way of life. Whereas CRC screening will moderate decline in mortality, and it may decrease the incidence of CRC through detection and removal of colorectal adenoma (early less invasive surgical treatment or endoscopic resection). Now that most of the early detection technologies such as fecal occult blood test, barium enema and electronic colonoscopy were low accuracy, high cost, invasive, non-compliance of patients, even serious complications such as intestinal bleeding and perforation and et al. So these methods is not suitable for screening. Therefore, exploration of a safe, reliable, specific, effective early CRC screening methods is the Urgent medical profession problem to be solved. CpG island hypermethylation of the tumor suppressor gene is the most common molecule change of CRC, and hypermethylation of the several genes is closely related to occurrence and development of CRC. Methylation changes often occurred in the early stage of CRC, which is easily detected in the faeces of patient. The characteristic and procedure of DNA methylation changes as tumor markers for the clinical diagnosis is superior to mutation detection. Thus, DNA methylation is closely correlated with early diagnosis, staging, treatment and prognosis of CRC and even can be used for large crowds screening, which has become a new research hot spots for early detection of CRC. In recent years, several studies have identified frequent promoter hypermethylation and silencing of the secreted frizzled-related protein 2 (SFRP2) genes in CRC, which is an early frequently event in colorectal tumorigenesis. This study is to investigate the feasibility of detecting hypermethylated SFRP2 gene in fecal DNA as a non-invasive screening tool for CRC by detecting faecal SFRP2 gene methylation status in patients with colorectal carcinoma and benign disease.
METHODS:
Methylation-specific PCR assay (MSP) was performed to analyze SFRP2 gene promoter methylation status in a blinded fashion in tumor tissues and in stool samples taken from 69 CRC patients preoperatively and at the 9th postoperative day, 34 patients with adenoma≥1 cm, 26 with hyperplastic polyp, and 30 endoscopically normal subjects. Simultaneously the relationship between hypermethylation of SFRP2 gene and clinicopathological features was analyzed.
RESULTS:
SFRP2 gene was hypermethylated in 91.3% (63/69) CRC, 79.4% (27/34) and 53.8% (14/26) adenoma and hyperplastic polyp tissues, and in 87.0% (60/69), 61.8% (21/34) and 42.3% (11/26) of corresponding fecal samples, respectively. In contrast, no methylated SFRP2 gene was detected in mucosal tissues of normal controls, while two cases of matched fecal samples from normal controls were detected with hypermethylated SFRP2. A significant decrease (P < 0.001) in the rate of hypermethylated SFRP2 gene was detected in the postoperative (8.7%, 6/69) fecal samples as compared with the preoperative fecal samples (87%, 60/69) of CRC patients. Moreover, no significant associations were observed between SFRP2 hypermethylation and clinicopathological features including sex, age, tumor stage, site, lymph node status and histological grade, etc.
CONCLUSION:
Hypermethylation of SFRP2 gene in fecal DNA is a novel molecular biomarker of CRC and carries a high potential for the remote detection of CRC and premalignant lesions as noninvasive screening method.
引文
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43.Kim HC, Roh SA, Ga IH, Kim JS, Yu CS, Kim JC. CpG island methylation as an early event during adenoma progression in carcinogenesis of sporadic colorectal cancer. J Gastroenterol Hepatol. 2005;20:1920-1926.
44.Wynter CV, Kambara T, Walsh MD, Leggett BA, Young J, Jass JR. DNA methylation patterns in adenomas from FAP, multiple adenoma and sporadic colorectal carcinoma patients. Int J Cancer. 2006;118:907-915.
45.Garber K. New gene discoveries may boost DNA stool testing for colorectal cancer. J Natl Cancer Inst. 2004;96:820-821.
46.McLoughlin RM, O'Morain CA. Colorectal cancer screening. World J Gastroenterol. 2006;12:674767-50.
47.Halligan S, Taylor SA. CT colonography: results and limitations. Eur J Radiol. 2007;61:400-408
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[3] Uren A, Reichsman F, Anest V, et al. Secreted frizzled-related protein-1 binds directly to Wingless and is a biphasic modulator of Wnt signaling.J Biol Chem, 2000, 275(6):4374-4382.
[4] Katoh M.Molecular Cloning and Characterization of MFRP,a Novel Gene Encoding a Membrane-Type Frizzled-Related Protein. Biochem Biophys Res Commun, 2001, 282(1):116-123.
[5]Jones SE, Jomary C. Secreted Frizzled-related proteins: searching for relationships and patterns. Bioessays, 2002,24(9):811-820.
[6] Lee JL, Lin CT, Chueh LL, et al. Autocrine/paracrine secreted Frizzled-related protein 2 induces cellular resistance to apoptosis: a possible mechanism of mammary tumorigenesis. J Biol Chem, 2004 , 279(15):14602-14609.
[7] Garcia-Hoyos M, Cantalapiedra D, Arroyo C, et al. Evaluation of SFRP1 as a candidate for human retinal dystrophies.Mol Vis, 2004,10:426-431.
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[10] Kawano Y, Kypta R. Secreted antagonists of the Wnt signalling pathway. J CellSci, 2003, 116(Pt 13):2627-2634.
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[12] Caldwell GM, Jones C, Gensberg K, et al. The Wnt antagonist SFRP1 in colorectal tumorigenesis. Cancer Res, 2004,64(3):883-888.
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[1] Fodde R, Smits R, Clevers H. APC, signal transduction and genetic instability in colorectal cancer. Nat. Rev. Cancer, 2001,1(1): 55-67.
[2] Bafico A, Gazit A, Pramila T, et al. Interaction of frizzled related protein(FRP) with Wnt ligands and the frizzled receptor suggests alternative mechanisms for FRP inhibition of Wnt signaling. J Biol Chem, 1999, 274(23):16180-16187.
[3] Uren A, Reichsman F, Anest V, et al. Secreted frizzled-related protein-1 binds directly to Wingless and is a biphasic modulator of Wnt signaling.J Biol Chem, 2000, 275(6):4374-4382.
[4] Katoh M.Molecular Cloning and Characterization of MFRP,a Novel Gene Encoding a Membrane-Type Frizzled-Related Protein. Biochem Biophys Res Commun, 2001, 282(1):116-123.
[5]Jones SE, Jomary C. Secreted Frizzled-related proteins: searching for relationships and patterns. Bioessays, 2002,24(9):811-820.
[6] Lee JL, Lin CT, Chueh LL, et al. Autocrine/paracrine secreted Frizzled-related protein 2 induces cellular resistance to apoptosis: a possible mechanism of mammary tumorigenesis. J Biol Chem, 2004 , 279(15):14602-14609.
[7] Garcia-Hoyos M, Cantalapiedra D, Arroyo C, et al. Evaluation of SFRP1 as a candidate for human retinal dystrophies.Mol Vis, 2004,10:426-431.
[8] Suzuki H, Gabrielson E, Chen W, et al. A genomic screen for genes upregulated by demethylation and histone deacety. lase inhibition in human colorectal cancer. Nat Genet,2002, 31(2):141-149.
[9] Suzuki H, Toyota M, Nojima M, et al. SFRP, a family of new colorectal tumor suppressor candidate genes. Nippon Rinsho, 2005, 63(4):707-719.
[10] Kawano Y, Kypta R. Secreted antagonists of the Wnt signalling pathway. J CellSci, 2003, 116(Pt 13):2627-2634.
[11] Suzuki H, Watkins DN, Jair KW, et al. Epigenetic inactivation of SFRP genes allows constitutive WNT signaling in colorectal cancer. Nat Genet, 2004 ,36(4):417-422.
[12] Caldwell GM, Jones C, Gensberg K, et al. The Wnt antagonist SFRP1 in colorectal tumorigenesis. Cancer Res, 2004,64(3):883-888.
[13] Morin PJ, Sparks AB, Korinek V, et al. Activation of beta-catenin-Tcf signaling in colon cancer by mutations in beta-catenin or APC. Science, 1997,275(5307):1787-1790.
[14] Feng Han Q, Zhao W, Bentel J, et al. Expression of SFRP-4 and beta-catenin in human colorectal carcinoma.Cancer Lett, 2006,231(1):129-137.
[15] Huiping C, Kristjansdottir S, Jonasson JG, et al. Alterations of E-cadherin and beta-catenin in gastric cancer.BMC Cancer, 2001,1:16.
[16] Mazieres J, He B, You L,et al. Wnt signaling in lung cancer. Cancer Lett, 2005,222(1):1-10.
[17] Sparks AB, Morin PJ, Vogelstein B, et al. Mutational analysis of the APC/β-catenin / Tcf pathway in colorectal cancer. Cancer Res, 1998, 58(6):1130-1134.
[18] Baylin SB, Herman JG. DNA hypermethylation in tumorigenesis: epigenetics joins genetics. Trends Genet, 2000,16(4): 168-174.
[19] Bafico A, Liu G, Goldin L, et al. An autocrine mechanism for constitutive Wnt pathway activation in human cancer cells. Cancer Cell, 2004,6(5): 497-506.
[20] Toyota M, Ahuja N, Ohe-Toyota M, et al. CpG island methylator phenotype in colorectal cancer. Proc Natl Acad Sci USA, 1999,96(15): 8681-8686.
[21] Gregorieff A, Clevers H. Wnt signaling in the intestinal epithelium: from endoderm to cancer. Genes Dev, 2005,19(8) :877-890.
[22] Traverso G, Shuber A, Levin B, et al. Detection of APC mutations in faecal DNA from patients with colorectal tumors. N Eugl J Med, 2002,346(5):311-320.
[23] Tagore KS, Lawson MJ, Yucaitis JA, et al. Sensitivity and Specificity of a stool DNA multitarget assay panel for the detection of advanced colorectal neoplasia. Clin colorectal Cancer, 2003,3(1): 47-53.
[24] Muller HM, Oberwalder M, Fiegl H, et al. Methylation changes in faecal DNA: a marker for colorectal cancer screening? Lancet, 2004,363(9417): 1283-1285.
[25] Davies RJ, Freeman A, Morris LS, et al. Analysis of minichromosome maintenance proteins as a novel method for detection of colorectal cancer in stool. Lancet, 2002,359(9321): 1917-1919.