Colorectal Cancer Screening: Stool DNA and Other Noninvasive Modalities

Colorectal cancer screening dates to the discovery of pre-cancerous adenomatous tissue. Screening modalities and guidelines directed at prevention and early detection have evolved and resulted in a significant decrease in the prevalence and mortality of colorectal cancer via direct visualization or using specific markers. Despite continued efforts and an overall reduction in deaths attributed to colorectal cancer over the last 25 years, colorectal cancer remains one of the most common causes of malignancy-associated deaths. In attempt to further reduce the prevalence of colorectal cancer and associated deaths, continued improvement in screening quality and adherence remains key. Noninvasive screening modalities are actively being explored. Identification of specific genetic alterations in the adenoma-cancer sequence allow for the study and development of noninvasive screening modalities beyond guaiac-based fecal occult blood testing which target specific alterations or a panel of alterations. The stool DNA test is the first noninvasive screening tool that targets both human hemoglobin and specific genetic alterations. In this review we discuss stool DNA and other commercially available noninvasive colorectal cancer screening modalities in addition to other targets which previously have been or are currently under study.     

Population Screening for Colorectal Cancer Means Getting FIT: The Past,Present, and Future of Colorectal Cancer Screening Using the Fecal Immunochemical Test for Hemoglobin (FIT)

Fecal immunochemical tests for hemoglobin (FIT) are changing the manner in which colorectal cancer (CRC) is screened. Although these tests are being performed worldwide, why is this test different from its predecessors? What evidence supports its adoption? How can this evidence best be used? This review addresses these questions and provides an understanding of FIT theory and practices to expedite international efforts to implement the use of FIT in CRC screening.     

粪便中p53与APC突变检测在大肠癌诊断中的意义

目的探讨在大肠癌患者粪便中检测p53、APC基因突变的可行性及其应用前景和意义。方法从36例大肠癌患者、10例大肠腺瘤患者以及30例正常对照者的粪便中分别提取DNA,应用PCR-SSCP法检测粪便中p53、APC基因突变情况。结果36例大肠癌患者粪便中p53及/或APC基因突变检出率为77.78%(19/36),二者突变率分别为52.78%(19/36)和36.11%(13/36);10例大肠腺瘤中p53基因突变检出率为0%,APC为20%;30例正常对照粪便中p53、APC基因突变检出率均为0%。p53的突变随大肠癌分化程度的降低而增高(P<0.05);APC基因突变与大肠癌组织学类型无关(P>0.05)。结论联合检测粪便中p53与APC突变在大肠癌诊断和筛查中有潜在的应用价值。     

Stool-based DNA testing, a new noninvasive method for colorectal cancer screening, the first report from Iran

AIM:To detect tumor-associated DNA changes in stool samples among Iranian patients with colorectal cancer(CRC)compared to healthy individuals using BAT-26,p16 hypermethylation and long DNA markers.METHODS:Stool DNA was isolated from 45 subjects including 25 CRC patients and 20 healthy individuals using a new,fast and easy extraction method.Long DNA associated with tumor was detected using polymerase chain reaction method.Microsatellite studies were performed utilizing denaturating polyacrylamide gel to determine the instability of BAT-26.Methylation status of p16 promoter was analyzed using methylation-specific PCR(MSP).RESULTS:The results showed a significant difference in existence of long DNA(16 in patients vs 1 in controls,P < 0.001)and p16(5 in patients vs none in controls,P = 0.043)in the stool samples of two groups.Long DNA was detected in 64% of CRC patients;whereas just one of the healthy individuals was positive for Long DNA.p16 methylation was found in 20% of patients and in none of healthy individuals.Instability of BAT-26 was not detected in any of stool samples.CONCLUSION:We could detect colorectal cancer related genetic alterations by analyzing stool DNA witha sensitivity of 64% and 20% and a specificity of 95% and 100% for Long DNA and p16 respectively.A non-invasive molecular stool-based DNA testing can provide a screening strategy in high-risk individuals.However,additional testing on more samples is necessary from Iranian subjects to determine the exact specificity and sensitivity of these markers.     

Hypermethylated SFRP2 gene in fecal DNA is a high potential biomarker for colorectal cancer noninvasive screening

AIM： To investigate the feasibility of detecting hypermethylated secreted frizzled-related protein 2 （SFRP2） gene in fecal DNA as a non-invasive screening tool for colorectal cancer （CRC）. METHODS： Fluorescence-based real-time PCR assay （MethyLight） 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.     

Detection of promoter hypermethylation of Wnt antagonist genes in fecal samples for diagnosis of early colorectal cancer

AIM: To investigate the feasibility of detecting aberrantly hypermethylated Wnt-antagonist gene promoters (SFRP2 and WIF-1) in fecal DNA as non-invasive biomarkers for early colorectal cancer (CRC).METHODS: The methylation-specific polymerase chain reaction assay was performed to blindly analyze the methylation status of SFRP2 and WIF-1 gene promoters in fecal samples from 48 subjects with CRC, 35 with adenomas, 32 with hyperplastic polyps and 30 endoscopically normal subjects. Additionally, we compared the diagnostic efficiency of measuring the hypermethylated SFRP2 and WIF-1 genes in the feces to the fecal occult blood test (FOBT) for the early detection of CRC.RESULTS: Hypermethylated SFRP2 was detected in the feces of 56.3% (27/48) of CRC cases, 51.4% (18/35) of adenoma cases and 12.5% (4/32) of patients with hyperplastic polyps. The hypermethylation of WIF-1 was detected in 60.4% (29/48), 45.7% (16/35) and 18.7% (6/32) of fecal samples from CRC, adenoma and hyperplastic polyp patients, respectively. At least one hypermethylated gene was detected in 81.3% (39/48) of CRC and 65.7% (23/35) of adenoma samples. In contrast, only a hypermethylated WIF-1 gene was detected in one case of normal fecal samples. Moreover, no significant associations were observed between SFPR2 and WIF-1 hypermethylation and clinicopathological features. Additionally, 81.8% of CRC cases diagnosed as Dukes A stage or advanced adenomas had at least one hypermethylated gene detected, while the detection rate with the FOBT was only 31.8% (P < 0.001).CONCLUSION: Hypermethylated SFRP2 and WIF-1 genes in fecal DNA are novel and promising molecular biomarkers that have great diagnostic potential for early CRC.     

Hypermethylation of <Emphasis Type="Italic">SFRP2</Emphasis> as a Potential Marker for Stool-Based Detection of Colorectal Cancer and Precancerous Lesions

DNA methylation is a key mechanism of colorectal carcinogenesis. Analysis of aberrantly methylation in stool DNA might provide a novel strategy for noninvasive detection of colorectal cancer (CRC). To explore the feasibility of this approach, we have assessed the methylation status of secreted frizzled-related protein gene 2 (SFRP2) in stool samples from patients with CRC with respect to a series of healthy individuals and patients with benign colorectal diseases, using methylation-specific polymerase chain reaction. Methylated SFRP2 occurs in 94.2%, 52.4%, 37.5%, and 16.7% of patients with CRC, adenomas, hyperplstic polyps, and ulcerative colitis, respectively. Of the 24 normal individuals, only 1 revealed methylated DNA. The pilot study revealed that aberrant methylated SFRP2 could be detected frequently in stools from patients with CRC and precancerous lesions. Methylation testing of fecal DNA may be a simple, promising, and noninvasive screening tool for colorectal neoplasia.     

Diagnostic value of stool DNA testing for multiple markers of colorectal cancer and advanced adenoma: A meta-analysis

BACKGROUND AND OBJECTIVES:

The diagnostic value of stool DNA (sDNA) testing for colorectal neoplasms remains controversial. To compensate for the lack of large-scale unbiased population studies, a meta-analysis was performed to evaluate the diagnostic value of sDNA testing for multiple markers of colorectal cancer (CRC) and advanced adenoma.

METHODS:

The PubMed, Science Direct, Biosis Review, Cochrane Library and Embase databases were systematically searched in January 2012 without time restriction. Meta-analysis was performed using a random-effects model using sensitivity, specificity, diagnostic OR (DOR), summary ROC curves, area under the curve (AUC), and 95% CIs as effect measures. Heterogeneity was measured using the χ² test and Q statistic; subgroup analysis was also conducted.

RESULTS:

A total of 20 studies comprising 5876 individuals were eligible. There was no heterogeneity for CRC, but adenoma and advanced adenoma harboured considerable heterogeneity influenced by risk classification and various detection markers. Stratification analysis according to risk classification showed that multiple markers had a high DOR for the high-risk subgroups of both CRC (sensitivity 0.759 [95% CI 0.711 to 0.804]; specificity 0.883 [95% CI 0.846 to 0.913]; AUC 0.906) and advanced adenoma (sensitivity 0.683 [95% CI 0.584 to 0.771]; specificity 0.918 [95% CI 0.866 to 0.954]; AUC 0.946) but not for the average-risk subgroups of either. In the methylation subgroup, sDNA testing had significantly higher DOR for CRC (sensitivity 0.753 [95% CI 0.685 to 0.812]; specificity 0.913 [95% CI 0.860 to 0.950]; AUC 0.918) and advanced adenoma (sensitivity 0.623 [95% CI 0.527 to 0.712]; specificity 0.926 [95% CI 0.882 to 0.958]; AUC 0.910) compared with the mutation subgroup. There was no significant heterogeneity among studies for subgroup analysis.

CONCLUSION:

sDNA testing for multiple markers had strong diagnostic significance for CRC and advanced adenoma in high-risk subjects. Methylation makers had more diagnostic value than mutation markers.     

Evaluation of Calgranulin B in Stools from the Patients with Colorectal Cancer

Purpose  Calprotectin (heterodimer of calgranulin A and B) has been previously studied as a candidate stool marker for detecting colorectal cancer. We assessed the clinical usefulness of calgranulin B as a stool marker for colorectal cancer in a pilot study of patients with colorectal cancer. Methods  We performed 2-DE-based proteomics to screen stool markers for colorectal cancer. We checked the calgranulin B in stools from 77 colorectal cancer patients and from 75 controls by western blot and enzyme-linked immunosorbent assay. We measured calgranulin A using the same methods, and stool hemoglobin by immunologic fecal occult blood test. Results  Fecal calgranulin A did not show any difference, but stool calgranulin B of colorectal cancer patients was significantly higher than controls [50.6 ng/mg stool protein (SD, 34.8) vs. 20.2 ng/mg stool protein (SD,24.0), respectively, P < 0.001). At the cut off level 24.4 ng/mg stool protein, the sensitivity was somewhat higher than fecal occult blood test (72.0 percent vs. 62.3 percent) but the specificity was much lower than fecal occult blood test (77.1 percent vs. 98.7 percent). Conclusions  Calgranulin B was increased in stools of colorectal cancer patients but our results suggest that colorectal cancer screening by determination of stool calgranulin B would not be better than conventional fecal occult blood test. This work was supported by research grants 0710670–1 (Yoo BC) and 0410063–3 (Lim SB) from the National Cancer Center, Korea. Poster presentation at the meeting of European Biomarkers Summit and Proteomics Europe, Amsterdam, The Netherlands, September 4 to 5, 2007.     

联合检测vimentin和SFRP2甲基化在大肠癌筛查中的应用评价

目的评价在粪便样本中联合检测vimentin和SFRP2甲基化来筛查大肠癌的性能。方法搜集合格的新鲜粪便标本95例,其中40例大肠癌患者、25例进展期腺瘤患者和30例结肠镜阴性的正常人,应用甲基化特异性PCR(MSP)法检测vimentin和SFRP2甲基化状态,并与单个基因甲基化检测和粪隐血试验(FOBT)的诊断性能相比较是否有统计学差异。结果大肠癌组中vimentin和SFRP2甲基化阳性检出率分别为55.0%(22/40)和70.0%(28/40);进展期腺瘤组中为48.0%(12/25)和64.0%(16/25);正常对照组中为6.7%(2/30)和0(0/30)。在病例组中两基因联合检测的敏感性为83.1%(54/65),明显高于vimentin的52.3%(34/65)和SFRP2的67.7%(44/65),更要高于FOBT的27.7%(18/65)(均P0.05)。而在正常对照组中两基因联合检测的特异性为93.3%(28/30),与vimentin的93.3%(28/30)和SFRP2的100%(30/30)以及FOBT的96.7%(29/30)相比均无明显差异(均P0.05)。结论在大肠癌筛查中,联合检测粪便中vimentin和SFRP2基因甲基化状态明显优于单个基因和粪便潜血试验,具有潜在的应用价值。     

检测粪便p53在胰腺癌诊断中应用的初步探讨

目的：本研究旨在探讨粪便p53基因突变检测方法在胰腺癌诊断中的潜在价值。方法：对连续就诊的胰腺癌62例患者的粪便，采用改进的两阶段酚－氯仿抽提和高浓度蛋白酶K消化法提取DNA。随后，采用PCR－SSCP方法检测粪便p53外显子5－8的突变情况。结果：以测定胰液p53突变率作为对照，粪便p53检测的敏感性为0．8，特异性为0．68，准确性为0．71，阳性预测值为0．36，阴性预测值为0．94。胰腺癌患者手术切除标本和粪便p53变检测结果一致率为63．6％（7／11），不一致率为36．4％（4／11），阳性预测值为0．33，阴性预测值为0．75。胰腺癌患者粪便p53突变率为37．1％（23．62），良性消化道疾病为12．8％（5／39），正常人为5．5％（3／55）。结论：粪便p53突变检测是一项具有潜在应用价值的对胰腺癌高危人群进行筛查的方法。     

Detection of aberrant methylation in fecal DNA as a molecular screening tool for colorectal cancer and precancerous lesions

AIM: To investigate the feasibility of detecting methylated fecal DNA as a screening tool for colorectal carcinoma (CRC) and precancerous lesions. METHODS: Methylated secreted frizzled-related protein gene 2 (SFRP2 ), hyperplastic polyposis protein gene (HPP1 ) and O6-methylguanine-DNA methyltransferase gene (MGMT) in stools from 52 patients with CRC, 35 patients with benign colorectal diseases and 24 normal individuals were analyzed using methylation-specific PCR. RESULTS: Methylated SFRP2, HPP1 and MGMT were detected in 94.2%, 71.2%, 48.1% of CRC patients and 52.4%, 57.1%, 28.6% of adenoma patients, respectively. The overall prevalence of fecal DNA with at least one methylated gene was 96.2% and 81.8% in patients with CRC and precancerous lesions, respectively. In contrast, only one of the 24 normal individuals revealed methylated DNA. These results indicated a 93.7% sensitivity and a 77.1% specificity of the assay for detecting CRC and precancerous lesions. CONCLUSION: Methylation testing of fecal DNA using a panel of epigenetic markers may be a simple and promising non-invasive screening method for CRC and precancerous lesions.     

Minnesota Colorectal Cancer Initiative: Successful Development and Implementation of a Community-Based Colorectal Cancer Registry

PURPOSE The aim of the Minnesota Colorectal Cancer Initiative is to implement risk-specific interventions to decrease colorectal cancer morbidity and mortality by 1) assisting clinicians to identify and educate individuals and families at high and increased risk for colorectal cancer; 2) providing professional and community education; 3) maintaining a database to evaluate the effectiveness of preventive intervention strategies; and 4) facilitating colorectal cancer research.METHODS Two physician groups and the University Cancer Center founded the Minnesota Colorectal Cancer Initiative as a not-for-profit organization. Health care organizations, pharmaceutical companies, a consulting firm, and other practice groups provide continuing financial and other support. A database registry, risk-assessment survey, and consent document were developed and then were approved by an institutional review board. A trial enrollment was conducted. Minnesota Colorectal Cancer Initiative services are available to the public. Participants are actively recruited through member organizations. Minnesota Colorectal Cancer Initiative assesses hereditary risk and will document family history in the medical record on request. A personally targeted reply letter reviews risk factors and recommends specific screening and surveillance strategies for participants and their family members, and when appropriate, provides information regarding genetic counseling and testing services. Minnesota Colorectal Cancer Initiative services are free to participants.RESULTS Since 1999, Minnesota Colorectal Cancer Initiative has sent individually tailored reply letters providing risk-specific information about colorectal cancer to 717 participants and more than 3200 of their first-degree and second-degree relatives. More than 200 families, previously unidentified as having histories suggestive of hereditary colorectal cancer (attenuated familial polyposis and hereditary nonpolyposis colorectal cancer), have been identified; genetic services were explained and recommended. A formal program evaluation confirmed that Minnesota Colorectal Cancer Initiative provides useful information and materials and promotes intrafamilial communication about colon cancer risk and recommendations.CONCLUSIONS Minnesota Colorectal Cancer Initiative is a model of effective collaboration between academic and community health care providers. A community-based registry is a unique way to identify and provide personal, risk-specific information to large numbers of people at increased or high risk for colorectal cancer.Poster presentation at the meeting of the American Society of Colon and Rectal Surgeons, June 21 to 26, 2003, New Orleans, Louisiana.     

实时荧光定量PCR和甲基化特异性PCR检测结直肠癌APC、MLH1基因甲基化状态

背景：启动子区甲基化致肿瘤抑制基因失活在结直肠癌的发生、发展中起重要作用,检测肿瘤相关基因的甲基化状态,可能为寻找新的结直肠癌诊断、预后相关标记物提供依据.目的：比较实时荧光定量PCR（FO-PCR）与甲基化特异性PCR（MSP）检测基因甲基化状态的差异.方法：以FQ-PCR检测66例结直肠癌组织和20例癌旁组织中与结直肠癌的发生、发展相关的抑癌基因APC和错配修复基因MLH1的甲基化状态,同时以MSP检测结直肠癌组织中APC基因的甲基化状态.结果：根据FQ-PCR结果,结直肠癌组织中APC、MLH1基因甲基化阳性率显著高于癌旁组织（48.5%和54.5%对0%和0%,P=0.000）.FQ-PCR和MSP可检出的甲基化阳性对照DNA最低浓度分别为0.015 ng/μl和1.5 ng/μl,两者对APC基因甲基化状态的检测结果差异有统计学意义（P＜0.05）.结论：在DNA甲基化的检测手段中,FQ-PCR的敏感度优于MSP.     

高分辨率溶解曲线分析与基因测序检测粪便DNA对大肠癌筛查作用的比较

目的 用基因测序评价高分辨率溶解曲线分析( high-resolution melting,HRM)检测粪便DNA的可靠性.方法 收集2009年3月～2009年9月在惠州市中心人民医院住院的39例大肠癌患者的新鲜粪便标本约1g,应用HRM法和基因测序法检测粪便中apc、k-ras、p53基因的突变情况,并对结果进行统计...     

Predicting the presence of adenomatous polyps during colonoscopy with National Cancer Institute Colorectal Cancer Risk-Assessment Tool

AIM To evaluate the National Cancer Institute(NCI)Colorectal Cancer(CRC)Risk Assessment Tool as a predictor for the presence of adenomatous polyps(AP) found during screening or surveillance colonoscopy.METHODS This is a retrospective single center observational study.We collected data of adenomatous polyps in each colonoscopy and then evaluated the lifetime CRC risk.We calculated the AP prevalence across risk score quintiles,odds ratios of the prevalence of AP across risk score quintiles,area under curves(AUCs)and Youden’s indexes to assess the optimal risk score cut off value for AP prevalence status.RESULTS The prevalence of AP gradually increased throughout the five risk score quintiles:i.e.,27.63%in the first and 51.35%in the fifth quintile.The odd ratios of AP prevalence in the fifth quintile compared to the first and second quintile were 2.76[confidence interval(CI):1.71-4.47]and 2.09(CI:1.32-3.30).The AUC for all patients was 0.62(CI:0.58-0.66).Youden’s Index indicated the optimal risk score cutoff value discriminating AP prevalence status was 3.60.CONCLUSION Patients with the higher NCI risk score have higher risk of AP and subsequent CRC;therefore,measures to increase the effectiveness of CRC detection in these patients include longer withdrawal time,early surveillance colonoscopy,and choosing flexible colonoscopy over other CRC screening modalities.     

Decreased Expression of Monocyte Chemoattractant Protein-1 Predicts Poor Prognosis Following Curative Resection of Colorectal Cancer

Purpose  The significance of monocyte chemoattractant protein-1 in colorectal cancer is not well understood. The aim of this study was to investigate the significance of monocyte chemoattractant protein-1 expression in colorectal cancer patients undergoing potentially curative surgery. Methods  We studied 101 colorectal cancer patients who underwent potentially curative surgery. The concentration of monocyte chemoattractant protein-1 in the tumor and normal mucosa were measured. The expression of monocyte chemoattractant protein-1 was also evaluated immunohistochemically. Results  The tissue concentration of monocyte chemoattractant protein-1 in the tumor was significantly higher than that in the normal mucosa. The decreased monocyte chemoattractant protein-1 cancer/normal ratio was associated with lymph node involvement and could predict poor prognosis. On univariate analysis, the decreased monocyte chemoattractant protein-1 ratio, carcinoembryonic antigen levels, and serosal invasion were the significant factors for poor prognosis. Multivariate analysis showed that monocyte chemoattractant protein-1 ratio was the only independent risk factor predictive of a poor prognosis. Immunohistochemically, monocyte chemoattractant protein-1 was expressed in the cytoplasm. Conclusion  The decreased monocyte chemoattractant protein-1 ratio was an independent factor predicting poor prognosis in patients undergoing potentially curative surgery. Monocyte chemoattractant protein-1 deficiency may present a new therapeutic approach for colorectal cancer.     

粪便DNA中MGMT、XAF1基因启动子甲基化联合检测在结直肠癌筛查中的应用研究

目的使用甲基化特异性PCR(MSP)方法检测粪便DNA中O6-甲基鸟嘌呤-DNA甲基转移酶(MGMT)、X染色体连锁凋亡抑制蛋白相关因子1(XAF1)基因启动子甲基化情况,并探讨其在结直肠肿瘤诊断中的意义。方法收集40例结直肠腺癌、40例腺瘤性息肉、52例正常对照住院患者粪便标本,使用试剂盒提取其粪便中肠道脱离细胞DNA,通过MSP方法检测其MGMT、XAF1基因启动子甲基化情况。结果 MGMT、XAF1基因启动子甲基化在结直肠腺癌中的阳性率分别为50.0%、55.9%;在腺瘤性息肉中的阳性率分别为42.1%、52.6%;二者联合检测在结直肠腺癌及腺瘤性息肉中的阳性率分别为73.5%、68.4%;特异性为52.0%。结直肠腺癌患者粪便中粪便潜血阳性率为35.3%,CEA阳性率为35.3%。结论通过试剂盒提取粪便DNA具有较高成功率;粪便DNA中MGMT、XAF1基因启动子甲基化状态用于检测结直肠腺癌及腺瘤性息肉具有较高的敏感性。检测粪便基因甲基化有望成为CRC高风险人群筛查的一个重要途径。