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Ma L, Gong J, Zhao M, Kong X, Gao P, Jiang Y, Liu Y, Feng X, Si S, Cao Y. A Novel Stool Methylation Test for the Non-Invasive Screening of Gastric and Colorectal Cancer. Front Oncol 2022; 12:860701. [PMID: 35419280 PMCID: PMC8995552 DOI: 10.3389/fonc.2022.860701] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2022] [Accepted: 03/02/2022] [Indexed: 11/13/2022] Open
Abstract
Background Because of poor compliance or low sensitivity, existing diagnostic approaches are unable to provide an efficient diagnosis of patients with gastric and colorectal cancer. Here, we developed the ColoCaller test, which simultaneously detects the methylation status of the SDC2, TFPI2, WIF1, and NDRG4 genes in stool DNA, to optimize the screening of gastric and colorectal cancer in high-risk populations. Methods A total of 217 stool samples from patients with gastrointestinal cancer and from patients with negative endoscopy were prospectively collected, complete with preoperative and postoperative clinical data from patients. The methylation of these samples was detected using ColoCaller, which was designed by selecting CpGs with a two-step screening strategy, and was interpreted using a prediction model built using libSVM to evaluate its clinical value for gastric and colorectal cancer screening. Results Compared to pathological diagnosis, the sensitivity and specificity of the ColoCaller test in 217 stool DNA samples were 95.56% and 91.86%, respectively, for colorectal cancer, and 67.5% and 97.81%, respectively, for gastric cancer. The detection limit was as low as 1% in 8 ng of DNA. Conclusion In this study, we developed and established a new test, ColoCaller, which can be used as a screening tool or as an auxiliary diagnostic approach in high-risk populations with gastric and colorectal cancer to promote timely diagnosis and treatment.
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Affiliation(s)
- Liang Ma
- Department of Clinical Laboratory, China-Japan Friendship Hospital, Beijing, China
| | - Jian Gong
- Department of Research and Development, Apexbio Biotechnology (Suzhou) Co., Ltd., Suzhou, China
| | - Meimei Zhao
- Department of Clinical Laboratory, China-Japan Friendship Hospital, Beijing, China
| | - Xiaomu Kong
- Department of Clinical Laboratory, China-Japan Friendship Hospital, Beijing, China
| | - Peng Gao
- Department of Clinical Laboratory, China-Japan Friendship Hospital, Beijing, China
| | - Yongwei Jiang
- Department of Clinical Laboratory, China-Japan Friendship Hospital, Beijing, China
| | - Yi Liu
- Department of Clinical Laboratory, China-Japan Friendship Hospital, Beijing, China
| | - Xiaoyan Feng
- Department of Research and Development, Apexbio Biotechnology (Suzhou) Co., Ltd., Suzhou, China
| | - Shuang Si
- Department of General Surgery, China-Japan Friendship Hospital, Beijing, China
| | - Yongtong Cao
- Department of Clinical Laboratory, China-Japan Friendship Hospital, Beijing, China
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2
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Jin S, Ye Q, Hong Y, Dai W, Zhang C, Liu W, Guo Y, Zhu D, Zhang Z, Chen S, Wang Y, Li D, Ma W, Yang Z, Li J, Zheng Z, Luan J, Wu X, Jiang F, Xu C, Ding C. A systematic evaluation of stool DNA preparation protocols for colorectal cancer screening via analysis of DNA methylation biomarkers. Clin Chem Lab Med 2020; 59:91-99. [PMID: 32673280 DOI: 10.1515/cclm-2020-0300] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/11/2020] [Accepted: 06/22/2020] [Indexed: 12/24/2022]
Abstract
Objectives Colorectal cancer (CRC) screening using stool samples is now in routine use where tumor DNA methylation analysis for leading markers such as NDRG4 and SDC2 is an integral part of the test. However, processing stool samples for reproducible and efficient extraction of human genomic DNA remains a bottleneck for further research into better biomarkers and assays. Methods We systematically evaluated several factors involved in the processing of stool samples and extraction of DNA. These factors include: stool processing (solid and homogenized samples), preparation of DNA from supernatant and pellets, and DNA extraction with column and magnetic beads-based methods. Furthermore, SDC2 and NDRG4 methylation levels were used to evaluate the clinical performance of the optimal protocol. Results The yield of total and human genomic DNA (hgDNA) was not reproducible when solid stool scraping is used, possibly due to sampling variations. More reproducible results were obtained from homogenized stool samples. Magnetic beads-based DNA extraction using the supernatant from the homogenized stool was chosen for further analysis due to better reproducibility, higher hgDNA yield, lower non-hgDNA background, and the potential for automation. With this protocol, a combination of SDC2 and NDRG4 methylation signals with a linear regression model achieved a sensitivity and specificity of 81.82 and 93.75%, respectively. Conclusions Through the systematic evaluation of different stool processing and DNA extraction methods, we established a reproducible protocol for analyzing tumor DNA methylation markers in stool samples for colorectal cancer screening.
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Affiliation(s)
- Shengnan Jin
- School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, PR China.,Key Laboratory of Laboratory Medicine, Ministry of Education, Wenzhou Medical University, Wenzhou, Zhejiang, PR China
| | - Qian Ye
- School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, PR China.,Key Laboratory of Laboratory Medicine, Ministry of Education, Wenzhou Medical University, Wenzhou, Zhejiang, PR China
| | - Yanping Hong
- School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, PR China.,Key Laboratory of Laboratory Medicine, Ministry of Education, Wenzhou Medical University, Wenzhou, Zhejiang, PR China
| | - Wenqing Dai
- School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, PR China.,Key Laboratory of Laboratory Medicine, Ministry of Education, Wenzhou Medical University, Wenzhou, Zhejiang, PR China
| | - Chengliang Zhang
- School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, PR China.,Key Laboratory of Laboratory Medicine, Ministry of Education, Wenzhou Medical University, Wenzhou, Zhejiang, PR China
| | - Weihao Liu
- School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, PR China.,Key Laboratory of Laboratory Medicine, Ministry of Education, Wenzhou Medical University, Wenzhou, Zhejiang, PR China
| | - Ying Guo
- School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, PR China.,Key Laboratory of Laboratory Medicine, Ministry of Education, Wenzhou Medical University, Wenzhou, Zhejiang, PR China
| | - Dewen Zhu
- School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, PR China.,Key Laboratory of Laboratory Medicine, Ministry of Education, Wenzhou Medical University, Wenzhou, Zhejiang, PR China
| | - Zhengzheng Zhang
- School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, PR China.,Key Laboratory of Laboratory Medicine, Ministry of Education, Wenzhou Medical University, Wenzhou, Zhejiang, PR China
| | - Shiliang Chen
- School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, PR China.,Key Laboratory of Laboratory Medicine, Ministry of Education, Wenzhou Medical University, Wenzhou, Zhejiang, PR China
| | - Yourong Wang
- School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, PR China.,Key Laboratory of Laboratory Medicine, Ministry of Education, Wenzhou Medical University, Wenzhou, Zhejiang, PR China
| | - Dandan Li
- School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, PR China.,Key Laboratory of Laboratory Medicine, Ministry of Education, Wenzhou Medical University, Wenzhou, Zhejiang, PR China
| | - Wen Ma
- School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, PR China.,Key Laboratory of Laboratory Medicine, Ministry of Education, Wenzhou Medical University, Wenzhou, Zhejiang, PR China
| | - Zhengquan Yang
- School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, PR China.,Key Laboratory of Laboratory Medicine, Ministry of Education, Wenzhou Medical University, Wenzhou, Zhejiang, PR China
| | - Jinlei Li
- Department of Colorectal Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, PR China
| | - Zhihai Zheng
- Department of Colorectal Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, PR China
| | - Ju Luan
- School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, PR China.,Key Laboratory of Laboratory Medicine, Ministry of Education, Wenzhou Medical University, Wenzhou, Zhejiang, PR China.,Innovation Biomedical Co., Ltd., Wenzhou, Zhejiang, PR China
| | - Xiaoli Wu
- Department of Gastroenterology, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, PR China
| | - Feizhao Jiang
- Department of Colorectal Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, PR China
| | - Chang Xu
- Department of Colorectal Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, PR China
| | - Chunming Ding
- School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, PR China.,Key Laboratory of Laboratory Medicine, Ministry of Education, Wenzhou Medical University, Wenzhou, Zhejiang, PR China
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Blood free-circulating DNA testing by highly sensitive methylation assay to diagnose colorectal neoplasias. Oncotarget 2018; 9:16974-16987. [PMID: 29682198 PMCID: PMC5908299 DOI: 10.18632/oncotarget.24768] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/2017] [Accepted: 02/27/2018] [Indexed: 12/18/2022] Open
Abstract
Although methylated TWIST1 is a biomarker of colorectal neoplasia, its detection from serum samples is very difficult by conventional bisulfite-based methylation assays. Therefore, we have developed a new methylation assay that enables counting of even one copy of a methylated gene in a small DNA sample amount without DNA bisulfite treatment. We performed this study to evaluate the sensitivity and specificity of serum DNA testing by the new methylation assay in combination with and without the fecal immunochemical test for hemoglobin for the detection of colorectal neoplasia. This study comprised 113 patients with colorectal neoplasia and 25 control individuals. For the new methylation assay, DNA was treated in two stages with methylation-sensitive restriction enzymes, followed by measurement of copy numbers of hTERT and methylated TWIST1 by multiplex droplet digital PCR. The fecal immunochemical test had a sensitivity of 8.0% for non-advanced adenoma, 24.3% for advanced adenoma, and 44.4% for colorectal cancer, and a specificity of 88.0%. The new assay had a sensitivity of 36.0% for non-advanced adenoma, 30.0% for advanced adenoma, and 44.4% for colorectal cancer, and a specificity of 92.0%. Combination of the both tests increased the sensitivity to 40.0%, 45.7%, and 72.2% for the detection of non-advanced adenoma, advanced adenoma, and colorectal cancer, respectively, and resulted in a specificity of 84.0%. Combination of both tests may provide an alternative screening strategy for colorectal neoplasia including potentially precancerous lesions and colorectal cancer.
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Suehiro Y, Zhang Y, Hashimoto S, Takami T, Higaki S, Shindo Y, Suzuki N, Hazama S, Oka M, Nagano H, Sakaida I, Yamasaki T. Highly sensitive faecal DNA testing of TWIST1 methylation in combination with faecal immunochemical test for haemoglobin is a promising marker for detection of colorectal neoplasia. Ann Clin Biochem 2017; 55:59-68. [DOI: 10.1177/0004563217691064] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
Background As TWIST1 methylation is specific to colorectal neoplasia, detection of TWIST1 methylation from faeces samples might be useful for colorectal neoplasia screening. However, because the content of human DNA in faeces is very small, it is very difficult to detect TWIST1 methylation by conventional bisulphite-based methylation assays. Therefore, we developed a new methylation assay without bisulphite treatment, the combined restriction digital PCR assay, and evaluated its sensitivity and specificity in combination with and without the faecal immunochemical test for haemoglobin for colorectal neoplasia detection from faeces samples. Methods For the combined restriction digital PCR assay, DNA was treated with three methylation-sensitive restriction enzymes and an exonuclease, followed by measurement of TWIST1 methylation level by droplet digital PCR. Faecal DNA testing and faecal immunochemical test for haemoglobin were performed on 109 patients with colorectal neoplasia and 10 control individuals. Results Basic performance testing showed that the combined restriction digital PCR assay enabled detection of 0.14% of the TWIST1 methylation level for the lymphocyte DNA. The combined restriction digital PCR assay from faeces samples had a sensitivity of 22.2% (95% confidence interval, 2.8–60.0%) for non-advanced adenoma, 47.1% (95% confidence interval, 23.0–72.2%) for advanced adenoma, and 33.7% (95% confidence interval, 23.7–45.0%) for colorectal cancer, and a specificity of 100.0%. Combination of faecal immunochemical test for haemoglobin and faecal combined restriction digital PCR assay increased sensitivity to 82.4% (95% confidence interval, 56.6–96.2%) for the detection of advanced adenoma. Conclusions We developed the combined restriction digital PCR assay, a possible highly sensitive methylation assay. Combination of faecal combined restriction digital PCR assay with faecal immunochemical test for haemoglobin may provide an alternative screening strategy for colorectal neoplasia, especially for potentially precancerous lesions.
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Affiliation(s)
- Yutaka Suehiro
- Department of Oncology and Laboratory Medicine, Yamaguchi University Graduate School of Medicine, Ube, Japan
| | - Yibo Zhang
- Department of Oncology and Laboratory Medicine, Yamaguchi University Graduate School of Medicine, Ube, Japan
| | - Shinichi Hashimoto
- Department of Gastroenterology and Hepatology, Yamaguchi University Graduate School of Medicine, Ube, Japan
| | - Taro Takami
- Department of Gastroenterology and Hepatology, Yamaguchi University Graduate School of Medicine, Ube, Japan
| | - Shingo Higaki
- Department of Gastroenterology, Sentohiru Hospital, Ube, Japan
| | - Yoshitaro Shindo
- Department of Gastroenterological, Breast and Endocrine Surgery, Yamaguchi University Graduate School of Medicine, Ube, Japan
| | - Nobuaki Suzuki
- Department of Gastroenterological, Breast and Endocrine Surgery, Yamaguchi University Graduate School of Medicine, Ube, Japan
| | - Shoichi Hazama
- Department of Translational Research and Developmental Therapeutics against Cancer, Yamaguchi University Graduate School of Medicine, Ube, Japan
| | - Masaaki Oka
- Department of Gastroenterological, Breast and Endocrine Surgery, Yamaguchi University Graduate School of Medicine, Ube, Japan
| | - Hiroaki Nagano
- Department of Gastroenterological, Breast and Endocrine Surgery, Yamaguchi University Graduate School of Medicine, Ube, Japan
| | - Isao Sakaida
- Department of Gastroenterology and Hepatology, Yamaguchi University Graduate School of Medicine, Ube, Japan
| | - Takahiro Yamasaki
- Department of Oncology and Laboratory Medicine, Yamaguchi University Graduate School of Medicine, Ube, Japan
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Iannone A, Losurdo G, Pricci M, Girardi B, Massaro A, Principi M, Barone M, Ierardi E, Di Leo A. Stool Investigations for Colorectal Cancer Screening: From Occult Blood Test to DNA Analysis. J Gastrointest Cancer 2016; 47:143-151. [PMID: 26922358 DOI: 10.1007/s12029-016-9810-z] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
PURPOSE We report an update of current methods for colorectal cancer (CRC) screening based on fecal sample analysis. METHODS A systematic review of the literature was performed in MEDLINE, EMBASE, and Science Direct electronic databases. RESULTS Blood in the stools is the first and most used strategy. Fecal occult blood test (FOBT) and fecal immunochemical test (FIT) are the main methods. Both are economic, easy to perform with high specificity, and low sensitivity. Based on CRC multi-step process with genetic and epigenetic alterations in large bowel cell DNA, single mutations or panels of alterations have been detected. These tests have the advantage of a marked improvement of the sensitivity when compared to fecal blood. However, high costs, poor availability, and correct choice of marker panel represent the major limits. A specific sDNA panel including aberrantly methylated BMP3 and NDRG4 promoter regions, mutant k-ras and β-actin (a reference gene for human DNA quantity), and an immunochemical assay for human hemoglobin has been recently approved by Food and Drug Administration. Novel promising biomarkers for CRC screening are represented by microRNAs (miRNAs), a group of 18-25 nucleotide non-coding RNA molecules that regulate gene expression. Reports on these fecal biomarkers are case-control studies, and each of them evaluates single miRNAs or multi-target panels. On the other hand, some fecal proteins have been studied as possible CRC screening markers, even though they demonstrated poor results. Finally, alterations of estrogen receptor-beta (i.e., dramatic reduction in the early stage of CRC) have been demonstrated in tissue samples. CONCLUSIONS Specific investigations are warranted in order to add further noninvasive markers to the panel of CRC screening tools.
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Affiliation(s)
- Andrea Iannone
- Section of Gastroenterology, Department of Emergency and Organ Transplantation, University of Bari, Bari, Italy
| | - Giuseppe Losurdo
- Section of Gastroenterology, Department of Emergency and Organ Transplantation, University of Bari, Bari, Italy
| | - Maria Pricci
- Section of Gastroenterology, Department of Emergency and Organ Transplantation, University of Bari, Bari, Italy
| | - Bruna Girardi
- Section of Gastroenterology, Department of Emergency and Organ Transplantation, University of Bari, Bari, Italy
| | - Antonio Massaro
- Section of Gastroenterology, Department of Emergency and Organ Transplantation, University of Bari, Bari, Italy
| | - Mariabeatrice Principi
- Section of Gastroenterology, Department of Emergency and Organ Transplantation, University of Bari, Bari, Italy
| | - Michele Barone
- Section of Gastroenterology, Department of Emergency and Organ Transplantation, University of Bari, Bari, Italy
| | - Enzo Ierardi
- Section of Gastroenterology, Department of Emergency and Organ Transplantation, University of Bari, Bari, Italy
| | - Alfredo Di Leo
- Section of Gastroenterology, Department of Emergency and Organ Transplantation, University of Bari, Bari, Italy.
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Mansour H. Cell-free nucleic acids as noninvasive biomarkers for colorectal cancer detection. Front Genet 2014; 5:182. [PMID: 25221563 PMCID: PMC4145725 DOI: 10.3389/fgene.2014.00182] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2014] [Accepted: 05/29/2014] [Indexed: 12/16/2022] Open
Abstract
Cell-free nucleic acids (CFNA) have been reported by several authors in blood, stool, and urine of patients with colorectal cancer (CRC). These genetic biomarkers can be an indication of neoplastic colorectal epithelial cells, and can thus potentially be used as noninvasive tests for the detection of the disease in CRC patients and monitor their staging, without the need to use heavier and invasive tools. In a number of test-trials, these genetic tests have shown the advantage of non-invasiveness, making them well accepted by most of the patients, without major side effects. They have also shown a promising sensitivity and specificity in the detection of malignant and premalignant neoplasms. Moreover, costs for performing such tests are very low. Several studies reported and confirmed the proof of the principle for these genetic tests for screening, diagnosis, and prognosis; the main challenge of translating this approach from research to clinical laboratory is the validation from large and long-term randomized trials to prove sustainable high sensitivity and specificity. In this paper, we present a review on the noninvasive genetics biomarkers for CRC detection described in the literature and the challenges that can be encountered for validation processes.
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Affiliation(s)
- Hicham Mansour
- Biosciences Core Laboratories, Research Department, King Abdullah University of Science and Technology Thuwal, Kingdom of Saudi Arabia
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7
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Diagnostic value of stool DNA testing for multiple markers of colorectal cancer and advanced adenoma: a meta-analysis. CANADIAN JOURNAL OF GASTROENTEROLOGY = JOURNAL CANADIEN DE GASTROENTEROLOGIE 2013; 27:467-75. [PMID: 23936877 DOI: 10.1155/2013/258030] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
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 χ(2) 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.
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Deng L, Qi Z, Zou B, Wu H, Huang H, Kajiyama T, Kambara H, Zhou G. Digital detection of multiple minority mutants in stool DNA for noninvasive colorectal cancer diagnosis. Anal Chem 2012; 84:5645-52. [PMID: 22715805 DOI: 10.1021/ac3008016] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Abstract
Somatic mutations in stool DNA are quite specific to colorectal cancer (CRC), but a method being able to detect the extraordinarily low amounts of mutants is challengeable in sensitivity. We proposed a hydrogel bead-array to digitally count CRC-specific mutants in stool at a low cost. At first, multiplex amplification of targets containing multiple mutation loci of interest is carried out by a target enriched multiplex PCR (Tem-PCR), yielding the templates qualified for emulsion PCR (emPCR). Then, after immobilizing the beads from emPCR on a glass surface, the incorporation of Cy3-dUTP into the mutant-specific probes, which are specifically hybridized with the amplified beads from emPCR, is used to color the beads coated with mutants. As all amplified beads are hybridized with the Cy5-labeled universal probe, a mutation rate is readily obtained by digitally counting the beads with different colors (yellow and red). A high specificity of the method is achieved by removing the mismatched probes in a bead-array with electrophoresis. The approach has been used to simultaneously detect 8 mutation loci within the APC, TP53, and KRAS genes in stools from eight CRC patients, and 50% of CRC patients were positively diagnosed; therefore, our method can be a potential tool for the noninvasive diagnosis of CRC.
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Affiliation(s)
- Lili Deng
- Department of Pharmacology, Jinling Hospital, Nanjing University School of Medicine, Nanjing, Jiangsu 210002, PR China
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9
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Improved recovery of exfoliated colonocytes from feces using newly developed immunomagnetic beads. Gastroenterol Res Pract 2008; 2008:605273. [PMID: 19125182 PMCID: PMC2606972 DOI: 10.1155/2008/605273] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/11/2008] [Accepted: 11/07/2008] [Indexed: 02/06/2023] Open
Abstract
We demonstrated the feasibility of a new methodology for isolating colonocytes from feces. To reduce costs and improve the recovery rate of colonocytes from feces, we attempted to develop new immunomagnetic beads. Several sizes of magnetic beads were prepared and tagged with a monoclonal antibody against EpCAM. We made several new monoclonal antibodies against EpCAM, and each monoclonal antibody was tagged to the magnetic beads. In the simulation, the most efficient recovery of HT-29 cells was obtained using the smallest size of beads. Also, beads tagged with a monoclonal antibody with a higher affinity against EpCAM had a higher recovery rate. Similar results were obtained when the smallest size of beads with the highest-affinity monoclonal antibody was applied to clinical samples. The newly developed immunomagnetic beads may be useful for isolating colorectal cancer cells from feces, enabling the cytological or molecular biological diagnosis of CRC.
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Stein J, Loitsch SM, Shastri Y. Nicht-invasive Diagnostik kolorektaler Tumore – Hat der Guaiac-Test ausgedient? / Non-invasive detection of colorectal cancer – do we still need the guaiac-based fecal occult blood test? LABORATORIUMSMEDIZIN 2008; 32:158-167. [DOI: 10.1515/jlm.2008.021] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 09/11/2023]
Abstract
Zusammenfassung
Aufgrund der leichten Handhabung und des Nachweises einer Mortalitätssenkung gilt der Nachweis von okkultem Blut (FOBT) im Stuhl derzeit als das am weitesten verbreitete Screeningverfahren für das kolorektale Karzinom. Als nachteilig erweisen sich allerdings eine unzureichende Sensitivität, insbesondere beim Nachweis früher Stadien und eine nach wie vor geringe Akzeptanz in der Bevölkerung. Vorläufige Daten zum Nachweis von Calprotectin oder der Tumor-M2-PK im Stuhl ließen bessere Screeningeigenschaften erwarten. Aber auch hier schränkt die geringe Sensitivität für frühe Vorstufen und unzureichende Spezifität mit zu erwartenden hohen Folgekosten die Tauglichkeit der Tests deutlich ein. Die kürzlich entwickelten immunologischen FOBTs (I-FOBT) erweisen sich als spezifischer und sensitiver. Sie beruhen auf dem Nachweis von humanem Hämoglobin mittels spezifischer Antikörper und sind somit unabhängig von diätetischen oder medikamentösen Faktoren, was zu einer deutlich besseren Akzeptanz führt. Sie gelten derzeit als kosteneffektivste Verfahren unter den nichtinvasiven Screeningmaßnahmen. Der Nachweis von Tumor-DNA im Stuhl eröffnet eine neue Ära zum frühzeitigen Nachweis kolorektaler Karzinome. Erste kleinere Studien weisen auf eine sehr gute Sensitivität dieser Verfahren hin. Sie lagen für kolorektale Karzinome zwischen 62–91% und für Adenome zwischen 26–73% bei mit 93–100% sehr guter Spezifität. Als nachteilig im Vergleich zu den derzeit verfügbaren Screeningtests erweisen sich allerdings die vergleichsweise hohen Kosten.
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Affiliation(s)
- Jürgen Stein
- Gastroenterologie, Proktologie, Diabetologie, Ernährungsmedizin, St. Elisabethen-Krankenhaus, Katharina-Kasper-Kliniken, Frankfurt/Main, Deutschland
| | - Stefan M. Loitsch
- Gastroenterologie, Proktologie, Diabetologie, Ernährungsmedizin, St. Elisabethen-Krankenhaus, Katharina-Kasper-Kliniken, Frankfurt/Main, Deutschland
| | - Yogesh Shastri
- Gastroenterologie, Proktologie, Diabetologie, Ernährungsmedizin, St. Elisabethen-Krankenhaus, Katharina-Kasper-Kliniken, Frankfurt/Main, Deutschland
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11
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Affiliation(s)
- Bernard Levin
- University of Texas M.D. Anderson Cancer Center, Houston, Texas 90033-0804, USA.
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12
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Matsushita H, Matsumura Y, Moriya Y, Akasu T, Fujita S, Yamamoto S, Onouchi S, Saito N, Sugito M, Ito M, Kozu T, Minowa T, Nomura S, Tsunoda H, Kakizoe T. A new method for isolating colonocytes from naturally evacuated feces and its clinical application to colorectal cancer diagnosis. Gastroenterology 2005; 129:1918-27. [PMID: 16344060 DOI: 10.1053/j.gastro.2005.10.007] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/24/2005] [Accepted: 08/31/2005] [Indexed: 02/06/2023]
Abstract
BACKGROUND & AIMS The early detection of colorectal cancer is desired because this cancer can be cured surgically if diagnosed early. The purpose of the present study was to determine the feasibility of a new methodology for isolating colonocytes from naturally evacuated feces, followed by cytology or molecular biology of the colonocytes to detect colorectal cancer originating from any part of the colorectum. METHODS Several simulation studies were conducted to establish the optimal methods for retrieving colonocytes from any portion of feces. Colonocytes exfoliated into feces, which had been retrieved from 116 patients with colorectal cancer and 83 healthy volunteers, were analyzed. Part of the exfoliated colonocytes was examined cytologically, whereas the remainder was subjected to DNA analysis. The extracted DNA was examined for mutations of the APC, K-ras, and p53 genes using direct sequence analysis and was also subjected to microsatellite instability (MSI) analysis. RESULTS In the DNA analysis, the overall sensitivity and specificity were 71% (82 of 116) of patients with colorectal cancer and 88% (73 of 83) of healthy volunteers. The sensitivity for Dukes A and B was 72% (44 of 61). Furthermore, the sensitivity for cancers on the right side of the colon was 57% (20 of 35). The detection rate for genetic alterations using our methodology was 86% (80 of 93) when the analysis was limited to cases in which genetic alterations were present in the cancer tissue. CONCLUSIONS We have developed a new methodology for isolating colonocytes from feces. The present study describes a promising procedure for future clinical evaluations and the early detection of colorectal cancers, including right-side colon cancer.
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Affiliation(s)
- Hisayuki Matsushita
- Investigative Treatment Division, Research Center for Innovative Oncology, National Cancer Center Hospital East, Kashiwa, Japan
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13
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Haug U, Brenner H. New stool tests for colorectal cancer screening: a systematic review focusing on performance characteristics and practicalness. Int J Cancer 2005; 117:169-76. [PMID: 15880368 DOI: 10.1002/ijc.21016] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
New stool tests may be promising tools for future colorectal cancer (CRC) screening. The aim of this review was to summarize current evidence of performance characteristics and practicalness in a population-based screening setting of recently developed stool tests. The MEDLINE database was searched for relevant articles published until July 2004. Studies were included if they comprised more than 10 cases and more than 10 controls. Details on study population, performance characteristics and stool collection procedure were taken into account. Overall, 29 studies, mostly retrospective, were included, investigating 17 different stool markers or marker combinations. Underlying study populations were very heterogeneous and mostly very small. Half of the studies reported sensitivity for adenomas in addition to sensitivity for CRC, and fewer than half reported sensitivity by tumor stage or location. Performance characteristics of stool tests varied to a large extent. For most DNA-based markers, specificity was about 95% or higher, but sensitivity was mostly low even for invasive CRC. More studies with larger sample sizes were done for protein-based markers, which typically had lower specificity. In most studies, stool samples were frozen within a rather short time period after defecation. While promising performance characteristics have been reported for some tests, more pervasive evidence from larger, prospectively designed studies, which also consider aspects of practicalness, e.g., the possibility of mailing the samples, is needed.
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Affiliation(s)
- Ulrike Haug
- Department of Epidemiology, German Centre for Research on Ageing, Heidelberg, Germany.
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14
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Whitney D, Skoletsky J, Moore K, Boynton K, Kann L, Brand R, Syngal S, Lawson M, Shuber A. Enhanced retrieval of DNA from human fecal samples results in improved performance of colorectal cancer screening test. J Mol Diagn 2005; 6:386-95. [PMID: 15507679 PMCID: PMC1876426 DOI: 10.1016/s1525-1578(10)60536-3] [Citation(s) in RCA: 61] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
Colorectal cancer accounts for more than 10% of all cancer deaths but is curable, if detected early. We reported previously on a stool-based screening test in which DNA from stool samples is subjected to genome analysis; sensitivity of the test has been limited in part by inefficiency of retrieving DNA from stool. Our aim was to test the impact of a new purification method that would increase the yield of human DNA from stool. DNA from 86 cancer and 100 non-cancer subjects (diagnosed by colonoscopy) were purified from stool with a new method for DNA recovery based on sequence-specific capture with acrylamide gel immobilized capture probes as well as with a previously developed magnetic bead-capture procedure. The new purification method gives an average 5.4-fold increase in the quantity of human DNA that can routinely be retrieved from fecal samples. The increased recovery of DNA corresponds with an increase in assay sensitivity from 53% (CI: 42 to 64%) to 70% (CI: 59 to 79%); P = 0.0005 (by McNemar's test), with no change in specificity. The newly developed sample preparation method mitigates a major problem in detecting rare cancer-associated genetic changes in heterogeneous clinical samples such as stool.
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Affiliation(s)
- Duncan Whitney
- Applied Research Group, EXACT Sciences Corporation, 100 Campus Drive, Marlborough, MA, USA
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15
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Abstract
When used appropriately, screening for colorectal cancer (CRC) can reduce disease-related morbidity and mortality. Current methods include fecal occult blood testing (FOBT), flexible sigmoidoscopy [FS], barium enema, and colonoscopy; all are cost-effective techniques. Unfortunately, offering an array of options has not increased screening utilization, which continues to lag behind that of other common cancers. Newer techniques, particularly virtual colonoscopy (VC) and stool DNA testing, may offer attractive alternatives for healthcare provider recommendation and patient use.
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Affiliation(s)
- Matthew Q Bromer
- Gastroenterology Division, Temple University Medical School, Philadelphia, PA, USA
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16
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Abstract
BACKGROUND Mass screening for colorectal cancer reduces mortality and, with recent advances in molecular genetics, molecular stool-based tests have produced promising results. This article reviews this innovation and discusses its clinical significance. METHODS Medline searches were used to identify recent key articles relating to stool-based testing. Further articles were obtained by manual scanning of the reference lists of identified papers. RESULTS Current screening methods are based on endoscopic, radiological and stool-based testing. Recent recognition of the adenoma-carcinoma sequence and pathophysiological studies of colonic epithelium have enabled tumour markers to be used in the screening setting. Non-invasive molecular stool testing has now been shown to have a high sensitivity and specificity. CONCLUSION Recent studies on molecular stool-based testing have shown higher sensitivity and specificity than earlier studies, but larger clinical trials are required. Laboratory methods are still undergoing research, with the aim of improving sensitivity to allow large-scale testing.
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Affiliation(s)
- T Mak
- Department of General Surgery, Manchester Royal Infirmary, Manchester, UK.
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Wan J, Zhang ZQ, You WD, Sun HK, Zhang JP, Wang YH, Fu YH. Detection of K-ras gene mutation in fecal samples from elderly large intestinal cancer patients and its diagnostic significance. World J Gastroenterol 2004; 10:743-6. [PMID: 14991952 PMCID: PMC4716923 DOI: 10.3748/wjg.v10.i5.743] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
AIM: To study the diagnostic significance of K-ras gene mutations in fecal samples from elderly patients with large intestinal cancer.
METHODS: DNA was extracted in the fecal and tissue samples from 23 large intestinal cancer patients, 20 colonic adenomatoid polypus patients and 20 healthy subjects. The K-ras gene mutations at the first and second bases of codon 12 were detected by the allele specific mismatch method.
RESULTS: The K-ras gene mutation was 56.52% (13/23) in the large intestinal cancer patients, which was notably higher than that in the normal subjects whose K-ras gene mutation was 5% (1/20) (Χ2 = 12.93, P < 0.001). There was no significant difference in comparison with that of colonic adenomatoid polypus patients whose K-ras gene mutation was 30% (6/12) (Χ2 = 3.05, P > 0.05). The K-ras gene mutation at the second base of codon 12 was 92.13% (12/13) in the large intestinal cancer patients. There was no significant difference between the detection rate of K-ras gene mutation in the fecal and tissue samples (Χ2 = 9.35, P < 0.01).
CONCLUSION: Our results indicate that detection of the K-ras gene mutations in fecal samples provides a non-invasive diagnostic method for the elderly large intestinal cancer patients. Its significance in the early diagnosis of large intestinal cancer awaits further studies.
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Affiliation(s)
- Jun Wan
- Department of Geriatric Gastroenterology, South Building of General Hospital of the Chinese PLA, Beijing, China.
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18
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Mizuno M, Mizuno M, Iwagaki N, Nasu J, Okazaki H, Yamamoto K, Okada H, Tsuji T, Fujita T, Shiratori Y. Testing of multiple samples increases the sensitivity of stool decay-accelerating factor test for the detection of colorectal cancer. Am J Gastroenterol 2003; 98:2550-5. [PMID: 14638362 DOI: 10.1111/j.1572-0241.2003.08672.x] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
OBJECTIVES We previously reported that the measurements of stool decay-accelerating factor (DAF), a membrane-bound, complement regulatory protein, may be valuable for the detection of colorectal cancer. Recently we have refined the immunoassay for stool DAF. In the present study, using the refined assay, we measured stool DAF concentrations in multiple samples from patients with colorectal cancer and in healthy controls to determine whether testing of multiple samples would increase the sensitivity of the stool DAF test. METHODS DAF was measured in three spontaneously passed stool samples from each of 100 patients with colorectal cancer and 100 control subjects without apparent colorectal disease. RESULTS The stool DAF concentrations in the patients with colorectal cancer (median 11.1 ng/g stool; interquartile range 2.9-32.7 ng/g) were significantly higher than concentrations in the subjects without colorectal diseases (median 1.6 ng/g stool; interquartile range 0.4-3.4 ng/g) (p<0.0001). Testing of two samples from each patient significantly increased the sensitivity (72%) of the stool DAF test without significantly decreasing its specificity (92%). The stool DAF test was positive in more than one half of patients with colorectal cancer at a relatively early TNM stage or with negative fecal occult blood test. CONCLUSIONS These findings suggest that stool DAF is a marker of colorectal cancer independent of fecal occult blood and testing of two samples increases the sensitivity of the stool DAF test. Measurement of stool DAF now seems worthy of further consideration as a noninvasive method for the detection of colorectal cancer.
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Affiliation(s)
- Masakatsu Mizuno
- Department of Medicine and Medical Science (Medicine 1), Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan
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Calistri D, Rengucci C, Bocchini R, Saragoni L, Zoli W, Amadori D. Fecal multiple molecular tests to detect colorectal cancer in stool. Clin Gastroenterol Hepatol 2003; 1:377-83. [PMID: 15017656 DOI: 10.1053/s1542-3565(03)00186-1] [Citation(s) in RCA: 57] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
BACKGROUND & AIMS Evaluation of molecular alterations in fecal DNA is a potential, noninvasive, alternative tool for the detection of colorectal cancer. We analyzed a large panel of molecular alterations involved in tumor transformation and progression to define their single diagnostic contribution in terms of sensitivity, cost, and time required to carry out the different tests. METHODS DNA was analyzed in stool from 38 healthy individuals and in paired stools and primary lesions from 56 patients with colorectal cancer. p53 exons 5-8, K-ras exons 1-2, four fragments of adenomatous polyposis coli (APC) exon 15, and 5 microsatellite loci were analyzed. Moreover, DNA amplification was evaluated for 4 exons of both p53 and APC. RESULTS K-ras (34%) and p53 (34%) mutations were the most frequent alterations in tumors, followed by microsatellite instability (13%) and APC mutations (13%). The most frequent event in stool was DNA amplification (51%), followed by alterations of K-ras (11%), p53 and microsatellite instability (6%), and APC (2%). K-ras and p53 gene mutations increased the capacity of DNA amplification to detect tumor cells by 8%. CONCLUSIONS K-ras and p53 gene mutations were the most frequent alterations observed in stool from patients with colorectal cancer, but DNA amplification was even more frequent, being present in more than half of patients. If these preliminary results are confirmed in a prospective study on a larger case series, this approach could be used for noninvasive colon cancer diagnosis in screening programs.
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Affiliation(s)
- Daniele Calistri
- Instituto Oncologico Romagnolo, Morgagni Hospital, Forli, Italy.
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Affiliation(s)
- C Pox
- Medizinische Klinik, Knappschaftskrankenhaus, Ruhr-Universität Bochum
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21
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Jubb AM, Quirke P, Oates AJ. DNA methylation, a biomarker for colorectal cancer: implications for screening and pathological utility. Ann N Y Acad Sci 2003; 983:251-67. [PMID: 12724230 DOI: 10.1111/j.1749-6632.2003.tb05980.x] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023]
Abstract
Currently up to one-third of colorectal cancer patients present with locally advanced or metastatic disease that precludes a surgical cure. Performance limitations and low uptake of current screening tools have fueled research to develop minimally invasive approaches that can detect early-stage neoplasms. The observation that altered DNA can be amplified from the stool or circulation has stimulated research on its use as a biomarker of occult neoplasia. De novo methylation of CpG islands 5' to certain tumor suppressor genes has been associated with epigenetic silencing. At certain loci this phenomenon is specific for neoplastic populations, and it is frequently detected at early stages in colorectal tumorigenesis. Accordingly, hypermethylation events have been proposed by researchers as ideal targets for the basis of a screening panel to detect peripheral tumor DNA. This critique reviews research findings on the use of epigenetic biomarkers in screening for occult neoplasia. In addition, the authors consider the pathological utility of epigenetic testing in refining tumor staging and predicting disease recurrence.
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Affiliation(s)
- Adrian M Jubb
- Academic Unit of Pathology, Leeds University, Leeds, UK.
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22
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Nishikawa T, Maemura K, Hirata I, Matsuse R, Morikawa H, Toshina K, Murano M, Hashimoto K, Nakagawa Y, Saitoh O, Uchida K, Katsu K. A simple method of detecting K-ras point mutations in stool samples for colorectal cancer screening using one-step polymerase chain reaction/restriction fragment length polymorphism analysis. Clin Chim Acta 2002; 318:107-12. [PMID: 11880119 DOI: 10.1016/s0009-8981(01)00806-3] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/09/2023]
Abstract
BACKGROUND We examined a technique for detecting point mutations of K-ras codon 12 in stool samples using one-step polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) analysis, in order to determine whether it could be used to screen for colorectal cancer. METHODS DNA was extracted from 200-mg stool specimens of 5 healthy controls and 31 colorectal cancer patients. A 107-base-pair fragment of exon 1 of K-ras was amplified by PCR using mismatched primers. PCR products were digested with Bst NI and analyzed by gel electrophoresis followed by silver staining. Specificity of one-step PCR/RFLP was examined by using synthetic oligonucleotides. The detection limit of K-ras codon 12 mutations was determined by using SW480 and HT29 cells. RESULTS The K-ras gene was successfully amplified from all healthy controls and colorectal cancer patients studied. Mutations of K-ras codon 12 were not detected in any of the healthy controls, but were identified in 13 (41.9%) of the 31 patients with colorectal cancer. Mutations were detectable in all six synthetic mutant DNAs, while none were detected among the wild type. The detection limit of this method was > or = 0.1%. CONCLUSIONS PCR/RFLP analysis could be used in mass screening for colorectal cancer, because it is highly specific, has a low detection limit, and is simpler than conventional methods for detecting genetic abnormalities.
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Affiliation(s)
- Takashi Nishikawa
- Second Department of Internal Medicine, Osaka Medical College, Takatsuki, Japan.
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23
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Traverso G, Shuber A, Levin B, Johnson C, Olsson L, Schoetz DJ, Hamilton SR, Boynton K, Kinzler KW, Vogelstein B. Detection of APC mutations in fecal DNA from patients with colorectal tumors. N Engl J Med 2002; 346:311-20. [PMID: 11821507 DOI: 10.1056/nejmoa012294] [Citation(s) in RCA: 248] [Impact Index Per Article: 10.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
BACKGROUND Noninvasive methods for detecting colorectal tumors have the potential to reduce morbidity and mortality from this disease. The mutations in the adenomatous polyposis coli (APC) gene that initiate colorectal tumors theoretically provide an optimal marker for detecting colorectal tumors. The purpose of our study was to determine the feasibility of detecting APC mutations in fecal DNA with the use of newly developed methods. METHODS We purified DNA from routinely collected stool samples and screened for APC mutations with the use of a novel approach called digital protein truncation. Many different mutations could potentially be identified in a sensitive and specific manner with this technique. RESULTS Stool samples from 28 patients with nonmetastatic colorectal cancers, 18 patients with adenomas that were at least 1 cm in diameter, and 28 control patients without neoplastic disease were studied. APC mutations were identified in 26 of the 46 patients with neoplasia (57 percent; 95 percent confidence interval, 41 to 71 percent) and in none of the 28 control patients (0 percent; 95 percent confidence interval, 0 to 12 percent; P<0.001). In the patients with positive tests, mutant APC genes made up 0.4 to 14.1 percent of all APC genes in the stool. CONCLUSIONS APC mutations can be detected in fecal DNA from patients with relatively early colorectal tumors. This feasibility study suggests a new approach for the early detection of colorectal neoplasms.
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Affiliation(s)
- Giovanni Traverso
- Graduate Program in Human Genetics, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Johns Hopkins School of Medicine, Baltimore, USA
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Atkin W, Martin JP. Stool DNA-based colorectal cancer detection: finding the needle in the haystack. J Natl Cancer Inst 2001; 93:798-9. [PMID: 11390522 DOI: 10.1093/jnci/93.11.798] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
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Ahlquist DA, Skoletsky JE, Boynton KA, Harrington JJ, Mahoney DW, Pierceall WE, Thibodeau SN, Shuber AP. Colorectal cancer screening by detection of altered human DNA in stool: feasibility of a multitarget assay panel. Gastroenterology 2000; 119:1219-27. [PMID: 11054379 DOI: 10.1053/gast.2000.19580] [Citation(s) in RCA: 352] [Impact Index Per Article: 14.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
BACKGROUND & AIMS Assay of altered DNA exfoliated into stool represents an intriguing approach to screen for colorectal neoplasia, but multiple markers must be targeted because of genetic heterogeneity. We explored the feasibility of a stool assay panel of selected DNA alterations in discriminating subjects with colorectal neoplasia from those without. METHODS Freezer-archived stools were analyzed in blinded fashion from 22 patients with colorectal cancer, 11 with adenomas > or =1 cm, and 28 with endoscopically normal colons. After isolation of human DNA from stool by sequence-specific hybrid capture, assay targets included point mutations at any of 15 sites on K-ras, p53, and APC genes; Bat-26, a microsatellite instability marker; and highly amplifiable DNA. RESULTS Analyzable human DNA was recovered from all stools. Sensitivity was 91% (95% confidence interval, 71%-99%) for cancer and 82% (48%-98%) for adenomas > or =1 cm with a specificity of 93% (76%-99%). Excluding K-ras from the panel, sensitivities for cancer were unchanged but decreased slightly for adenomas to 73% (39%-94%), while specificity increased to 100% (88%-100%). CONCLUSIONS Assay of altered DNA holds promise as a stool screening approach for colorectal neoplasia. Larger clinical investigations are indicated.
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Affiliation(s)
- D A Ahlquist
- Division of Gastroenterology and Hepatology, Department of Biostatistics, and Division of Molecular Genetics, Mayo Clinic, Rochester, Minnesota 55905, USA.
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Lev Z, Kislitsin D, Rennert G, Lerner A. Utilization of K-ras mutations identified in stool DNA for the early detection of colorectal cancer. JOURNAL OF CELLULAR BIOCHEMISTRY. SUPPLEMENT 2000; 34:35-9. [PMID: 10762013 DOI: 10.1002/(sici)1097-4644(2000)77:34+<35::aid-jcb8>3.0.co;2-w] [Citation(s) in RCA: 23] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
Colorectal cancer is one of the most common malignancies in the western world. About 60,000 Americans die of colorectal cancer each year. The annual incidence rate in Israel is 40 per 100,000 persons, namely a total of 2,000 new cases each year. An important step in the progression of colorectal cancer includes induction of activating mutations in the proto-oncogene K-ras. The mutations in K-ras appear early during tumorigenesis, at the intermediate adenoma stage, and thus can be used as a biomarker for early detection in about 40% of colonic tumors. A large yet unknown number of mutated cells are shed from the developing tumor during its progression. Indeed, K-ras mutations were detected in DNA isolated from stool obtained from symptomatic and asymptomatic patients with colorectal cancer, suggesting a novel approach for a noninvasive screening procedure. However, severe difficulties in obtaining reproducible yields of amplifiable DNA from stool, and usage of nonquantitative, time-consuming procedures, hampered further progress in the utilization of K-ras mutations for the early detection of colorectal cancer. Apparently a novel protocol is required that provides reproducible output of amplifiable DNA from small amounts of stool, detects if K-ras mutated DNA is present, and determines the quantity of K-ras mutated cells in the stool sample. In addition, this protocol should be simple, robotics compatible, and thus suitable for cost-effective, large-scale mutation screening. Molecular assays for detecting K-ras mutations and additional biomarkers in stool DNA promise to be highly sensitive, specific, and cost-effective. As such they should be very effective when used in chemoprevention studies and screening protocols for colorectal cancer.
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Affiliation(s)
- Z Lev
- Department of Biology, Technion-Israel Institute of Technology, Haifa. mailto:
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Arber N, Shapira I, Ratan J, Stern B, Hibshoosh H, Moshkowitz M, Gammon M, Fabian I, Halpern Z. Activation of c-K-ras mutations in human gastrointestinal tumors. Gastroenterology 2000; 118:1045-50. [PMID: 10833479 DOI: 10.1016/s0016-5085(00)70357-x] [Citation(s) in RCA: 81] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
BACKGROUND & AIMS Ras genes are the most frequently detected oncogenes in human malignancies. Data regarding the frequency of c-K-ras mutations in esophageal, gastric, and small bowel tumors are limited and controversial. METHODS DNA was extracted from 262 formalin-fixed, paraffin-embedded sections of gastrointestinal samples and tumors, including Barrett's esophagus, esophageal squamous cell carcinomas and adenocarcinomas, and small and large bowel adenomas and adenocarcinomas. The presence of c-K-ras codon 12 mutations was determined using a nonradioactive polymerase chain reaction-based restriction fragment length polymorphism assay. RESULTS c-K-ras mutations were detected in 1 of 39 (2%) patients with Barrett's esophagus, 1 of 21 (5%) adenocarcinomas, 0 of 27 squamous cell carcinomas of the esophagus, and 1 of 32 (3%) gastric adenocarcinomas. It was also present in 8 of 20 (40%) and 10 of 28 (36%) small bowel adenomas and adenocarcinomas, respectively. Similar numbers were observed in 10 of 25 (40%) large bowel adenomas and 11 of 30 adenocarcinomas (37%). Mutations were not associated with age, gender, histology, grade, stage, location, or mortality. CONCLUSIONS The frequency of codon 12 c-K-ras mutations in small and large bowel tumors is approximately 10-fold higher than that of tumors in the upper gastrointestinal tract.
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Affiliation(s)
- N Arber
- GI Oncology Unit, Tel-Aviv University, Tel Aviv, Israel.
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Notarnicola M, Cavallini A, Cardone R, Pezzolla F, Demma I, Di Leo A. K-ras and p53 mutations in DNA extracted from colonic epithelial cells exfoliated in faeces of patients with colorectal cancer. Dig Liver Dis 2000; 32:131-6. [PMID: 10975788 DOI: 10.1016/s1590-8658(00)80400-4] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
BACKGROUND Exfoliated colonic epithelial cells in faeces provide a source of human DNA which may be analysed for the presence of tumour-induced modification. AIM In the present study we investigated K-ras and p53 mutations in faeces of patients with colorectal carcinoma, to verify whether analysis of these mutations might identify a high percentage of patients with colorectal cancer. PATIENTS AND METHODS Faeces, tumour and normal mucosa samples were taken from 26 patients. Polymerase chain reaction amplification and restriction enzyme analysis were performed to detect K-ras mutations; p53 gene mutations were identified by using polymerase chain reaction amplification and single strand conformation polymorphism. RESULTS We were able to amplify the K-ras gene and exons 5-9 of the p53 gene in 100% of the faecal samples studied. K-ras and p53 gene mutations were detected in faeces in 26.9% and 50% of the cases, respectively. The two mutations were present together in 5 out of 26 patients. There was full agreement between the K-ras and p53 pattern observed in faecal DNA and that in tumour tissue DNA. CONCLUSIONS Application of K-ras and p53 mutation gene analysis in the faeces may have clinical applications in the future. Since this genetic analysis is able to detect only 57.7% of patients with colorectal cancer, the study of other genes involved in colorectal carcinogenesis is necessary.
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Affiliation(s)
- M Notarnicola
- Laboratory of Biochemistry, IRCCS Scientific Institute for Digestive Diseases, S. de Bellis Castellana G., BA, Italy
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Puig P, Urgell E, Capellá G, Sancho FJ, Pujol J, Boadas J, Farré A, Lluís F, González-Sastre F, Mora J. A highly sensitive method for K-ras mutation detection is useful in diagnosis of gastrointestinal cancer. Int J Cancer 2000; 85:73-7. [PMID: 10585586 DOI: 10.1002/(sici)1097-0215(20000101)85:1<73::aid-ijc13>3.0.co;2-#] [Citation(s) in RCA: 40] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022]
Abstract
Detection of molecular features such as K-ras mutations has been used to evaluate potential tumour markers in a wide variety of clinical samples. Here we have applied a recently developed highly sensitive method for detection of K-ras codon 12 mutations to colorectal and pancreatic cancer diagnosis. We analysed 67 faecal samples from patients undergoing diagnostic colonoscopy under suspicion of colorectal cancer. PCR products were obtained in 62 of 67 (93%) faecal samples. Mutations were detected in exfoliated cells in 6 of 22 (27%) of the adenomas and in 6 of 11 (55%) of adenocarcinomas. No false positives were observed. Agreement between faecal samples and corresponding tissues was 100% for adenocarcinomas and 65% for adenomas. Mutations were also analysed in 61 pancreatic fine-needle aspirates. Mutations were detected in 36 of 45 (80%) of the pancreatic aspirates diagnosed as pancreatic cancer without false positives. Our findings suggest that, when colorectal cancer is suspected, detection of K-ras codon 12 mutations in faecal samples using this new method is specific for colorectal tumours. Additionally, this technique is a good alternative for evaluation of pancreatic masses.
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Affiliation(s)
- P Puig
- Department of Clinical Biochemistry, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain
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Salama SA, Serrana M, Au WW. Biomonitoring using accessible human cells for exposure and health risk assessment. Mutat Res 1999; 436:99-112. [PMID: 9878700 DOI: 10.1016/s1383-5742(98)00021-0] [Citation(s) in RCA: 43] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
A major goal for genetic toxicologist is to provide precise information on exposure and health risk assessment for effective prevention of health problems. A frequently used approach for population study has been to utilize readily available blood cells (lymphocytes and red blood cells) as sentinel cell types to detect biological effects from exposure and to provide early warning signals for health risk. However, such approach still cannot be used reliably for developing strategies in risk assessment and disease prevention. It is possible that other available cell types which are more representative of the target cells for disease may be used to overcome the deficiency. In this report, the use of non-blood cells for biomonitoring is briefly reviewed. Their usefulness in certain exposure condition is highlighted and their effectiveness in documenting exposure compared with other cell types such as the traditional blood cells is presented. It is obvious that the decision in using these non-blood cells in biomonitoring is based on the exposure condition and the experimental design. Nevertheless, monitoring studies using non-blood cells should be encouraged with emphasis on providing dose-response information, comparative response with other cell types and effectiveness for health risk assessment.
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Affiliation(s)
- S A Salama
- Department of Preventive Medicine and Community Health, The University of Texas Medical Branch, Galveston, TX 77555-1110, USA
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Ward R, Hawkins N, O'Grady R, Sheehan C, O'Connor T, Impey H, Roberts N, Fuery C, Todd A. Restriction endonuclease-mediated selective polymerase chain reaction: a novel assay for the detection of K-ras mutations in clinical samples. THE AMERICAN JOURNAL OF PATHOLOGY 1998; 153:373-9. [PMID: 9708798 PMCID: PMC1852993 DOI: 10.1016/s0002-9440(10)65581-2] [Citation(s) in RCA: 62] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/27/2023]
Abstract
The enriched polymerase chain reaction (PCR) assay has been used extensively in the detection of ras gene mutations in many types of human malignancies. Although it is very sensitive, it has a number of features that limit its use in the routine diagnostic laboratory. The aim of this study was to develop a novel enriched PCR strategy, in which the concurrent activity of the restriction enzyme BstNI and Taq polymerase allowed the amplification of mutant K-ras while inhibiting the formation of wild-type product. This restriction endonuclease-mediated selective PCR assay uses three sets of primers, together with BstNI, in the reaction mix, and the amplification products are analyzed by gel electrophoresis. The reliability of the restriction endonuclease-mediated selective PCR assay to detect activated K-ras was determined in a variety of clinical samples, including 139 fresh colorectal carcinomas and 113 paraffin-embedded blocks from 80 separate tumors of the colon and rectum, pancreas, breast, or kidney. Codon 12 mutations of the K-ras oncogene were identified in DNA from both fresh and paraffin-embedded tumors in a rapid, sensitive, and reproducible manner. Mutations were detected in 33 (24%) of the fresh colorectal cancers and 16 (20%) of the paraffin-embedded tumors. These results were 97% concordant in cases in which paraffin blocks and fresh specimens from the same tumor were available for analysis. We conclude that restriction endonuclease-mediated selective PCR is a sensitive, rapid, and robust assay for the detection of point mutations in a variety of clinical samples. Importantly, there is no need for manipulation of the sample once the PCR has been set up, and therefore, the chance of contamination is significantly reduced. In contrast to previous assays, restriction endonuclease-mediated selective PCR is not labor intensive, and its format is suitable for use in routine diagnostic laboratory.
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Affiliation(s)
- R Ward
- Department of Medical Oncology, St. Vincent's Hospital, Sydney, New South Wales, Australia.
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Ratto C, Sofo L, Ippoliti M, Merico M, Doglietto GB, Crucitti F. Prognostic factors in colorectal cancer. Literature review for clinical application. Dis Colon Rectum 1998; 41:1033-49. [PMID: 9715162 DOI: 10.1007/bf02237397] [Citation(s) in RCA: 110] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
PURPOSE Identification of prognostic factors is a primary basis for planning the treatment and predicting the outcome of patients with colorectal cancer. Reviewing studies from the literature performed using univariate and multivariate analyses and their own study, the authors critically discuss the prognostic value of the clinicopathologic parameters of the tumor. METHODS Among 853 patients with colorectal tumors seen at the Department of Clinical Surgery of the Catholic University of Rome, Italy, 690 cases that were curatively resected the study. Overall survival rate, related to the clinicopathologic variables, was calculated, and univariate and multivariate analyses were performed. RESULTS Five-year and ten-year overall survival rates were 70 and 55 percent, respectively. Univariate and multivariate analyses showed that node involvement, distant metastases, bowel obstruction, and patient gender are factors independently related to outcome. CONCLUSIONS Data from the literature and the present study suggest that only a few clinical parameters, particularly bowel obstruction, and some pathologic factors (tumor stage, vessels invasion, and tumor ploidy) are related to patient survival rate and are the most reliable prognostic criteria. In prospective clinical studies, any other new pathologic or molecular factors should be matched with these parameters to confirm their value in outcome prediction.
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Affiliation(s)
- C Ratto
- Department of Clinica Chirurgica, Catholic University, Rome, Italy
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