Detection of Hypermethylated Vimentin in Urine of Patients with Colorectal Cancer
2012; Elsevier BV; Volume: 14; Issue: 2 Linguagem: Inglês
10.1016/j.jmoldx.2011.12.003
ISSN1943-7811
AutoresBenjamin P. Song, Surbhi Jain, Selena Y. Lin, Quan Chen, Timothy M. Block, Wei Song, Dean E. Brenner, Ying‐Hsiu Su,
Tópico(s)Immunotherapy and Immune Responses
ResumoWe demonstrated previously that urine contains low-molecular-weight (LMW) (<300 bp), circulation-derived DNA that can be used to detect cancer-specific mutations if a tumor is present. The goal of this study was to develop an assay to detect the colorectal cancer (CRC)–associated, circulation-derived, epigenetic DNA marker hypermethylated vimentin gene (mVIM) in the urine of patients with CRC. An artificial 18-nucleotide DNA sequence was tagged at the 5′ end of the primers of the first PCR cycle to increase the amplicon size, which was then integrated into the primers of the second PCR cycle. A quantitative MethyLight PCR-based assay targeting a 39-nucleotide template was developed and used to quantify mVIM in CRC tissues and matched urine samples. mVIM was detected in 75% of LMW urine DNA samples from patients with CRC (n = 20) and in 10% of urine samples of control subjects with no known neoplasia (n = 20); 12 of 17 LMW urine DNA samples (71%) but only 2 of 17 high-molecular-weight urine DNA samples (12%) from patients with mVIM-positive tissues contained detectable mVIM, suggesting that the mVIM detected in LMW urine DNA is derived from the circulation. The detection of mVIM in urine was significantly associated with CRC compared with controls (P < 0.0001, by Fisher's exact test). A potential urine test for CRC screening using epigenetic markers is discussed. We demonstrated previously that urine contains low-molecular-weight (LMW) (<300 bp), circulation-derived DNA that can be used to detect cancer-specific mutations if a tumor is present. The goal of this study was to develop an assay to detect the colorectal cancer (CRC)–associated, circulation-derived, epigenetic DNA marker hypermethylated vimentin gene (mVIM) in the urine of patients with CRC. An artificial 18-nucleotide DNA sequence was tagged at the 5′ end of the primers of the first PCR cycle to increase the amplicon size, which was then integrated into the primers of the second PCR cycle. A quantitative MethyLight PCR-based assay targeting a 39-nucleotide template was developed and used to quantify mVIM in CRC tissues and matched urine samples. mVIM was detected in 75% of LMW urine DNA samples from patients with CRC (n = 20) and in 10% of urine samples of control subjects with no known neoplasia (n = 20); 12 of 17 LMW urine DNA samples (71%) but only 2 of 17 high-molecular-weight urine DNA samples (12%) from patients with mVIM-positive tissues contained detectable mVIM, suggesting that the mVIM detected in LMW urine DNA is derived from the circulation. The detection of mVIM in urine was significantly associated with CRC compared with controls (P < 0.0001, by Fisher's exact test). A potential urine test for CRC screening using epigenetic markers is discussed. Colorectal cancer (CRC) remains the second leading cause of cancer deaths in the United States (>49,380 projected deaths in 2011)1Siegel R. Ward E. Brawley O. Jemal A. Cancer statistics, 2011: the impact of eliminating socioeconomic and racial disparities on premature cancer deaths.CA Cancer J Clin. 2011; 61: 212-236Crossref PubMed Scopus (3846) Google Scholar despite the availability of sensitive screening tests, such as colonoscopy. The inconvenience of the test and the risks involved contribute to the low adherence rate (40%) in US adults. The noninvasive fecal occult blood test is also available, but its sensitivity is low (∼30%). Fecal DNA tests for CRC screening have been extensively studied and gave encouraging results (up to 90% sensitivity) with open-labeled study participants.2Dong S.M. Traverso G. Johnson C. Geng L. Favis R. Boynton K. Hibi K. Goodman S.N. D'Allessio M. Paty P. Hamilton S.R. Sidransky D. Barany F. Levin B. Shuber A. Kinzler K.W. Vogelstein B. Jen J. Detecting colorectal cancer in stool with the use of multiple genetic targets.J Natl Cancer Inst. 2001; 93: 858-865Crossref PubMed Scopus (326) Google Scholar, 3Osborn N.K. Ahlquist D.A. Stool screening for colorectal cancer: molecular approaches.Gastroenterology. 2005; 128: 192-206Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 4Syngal S. Stoffel E. Chung D. Willett C. Schoetz D. Schroy P. Jagadeesh D. Morel K. Ross M. Detection of stool DNA mutations before and after treatment of colorectal neoplasia.Cancer. 2006; 106: 277-283Crossref PubMed Scopus (43) Google Scholar, 5Ahlquist D.A. Skoletsky J.E. Boynton K. Harrington J.J. Mahoney D.W. Pierceall W.E. Thibodeau S.N. Shuber A.P. Colorectal cancer screening by detection of altered human DNA in stool: feasibility of a multitarget assay panel.Gastroenterology. 2000; 119: 1219-1227Abstract Full Text Full Text PDF PubMed Scopus (503) Google Scholar, 6Traverso G. Shuber A. Levin B. Johnson C. Olsson L. Schoetz Jr., D.J. Hamilton S.R. Boynton K. Kinzler K.W. Vogelstein B. Detection of APC mutations in fecal DNA from patients with colorectal tumors.N Engl J Med. 2002; 346: 311-320Crossref PubMed Scopus (295) Google Scholar However, the sensitivity to detect CRC fell to ≤52% in large multicenter validation studies in an average-risk population7Imperiale T.F. Ransohoff D.F. Itzkowitz S.H. Turnbull B.A. Ross M.E. Colorectal Cancer Study GroupFecal DNA versus fecal occult blood for colorectal-cancer screening in an average-risk population.N Engl J Med. 2004; 351: 2704-2714Crossref PubMed Scopus (707) Google Scholar, 8Ahlquist D.A. Sargent D.J. Loprinzi C.L. Levin T.R. Rex D.K. Ahnen D.J. Knigge K. Lance M.P. Burgart L.J. Hamilton S.R. Allison J.E. Lawson M.J. Devens M.E. Harrington J.J. Hillman S.L. Stool DNA and occult blood testing for screen detection of colorectal neoplasia.Ann Intern Med. 2008; 149: 441-450Crossref PubMed Scopus (247) Google Scholar and as reviewed by Bonanno et al9Bonanno E. Rulli F. Galatà G. Pucci S. Sesti F. Farinon A.M. Spagnoli L.G. Stool test for colorectal cancer screening: what is going on.Surg Oncol. 2007; 16: 43-45Abstract Full Text Full Text PDF Scopus (6) Google Scholar and Levin et al.10Levin B. Lieberman D.A. McFarland B. Andrews K.S. Brooks D. Bond J. Dash C. Giardiello F.M. Glick S. Johnson D. Johnson C.D. Levin T.R. Pickhardt P.J. Rex D.K. Smith R.A. Thorson A. Winawer S.J. American Cancer Society Colorectal Cancer Advisory Group; US Multi-Society Task Force; American College of Radiology Colon Cancer CommitteeScreening and surveillance for the early detection of colorectal cancer and adenomatous polyps, 2008: a joint guideline from the American Cancer Society, the US Multi-Society Task Force on Colorectal Cancer, and the American College of Radiology.Gastroenterology. 2008; 134: 1570-1595Abstract Full Text Full Text PDF PubMed Scopus (1728) Google Scholar This result could be due to the massive contamination from bacterial DNA in stool.11Klaassen C.H.W. Jeunink M.A.F. Prinsen C.F.M. Ruers T.J.M. Tan A.C.I.T.L. Strobbe L.J.A. Thunnissen F.B.J.M. Quantification of human DNA in feces as a diagnostic test for the presence of colorectal cancer.Clin Chem. 2003; 49: 1185-1187Crossref PubMed Scopus (67) Google Scholar, 12Diehl F. Schmidt K. Durkee K.H. Moore K.J. Goodman S.N. Shuber A.P. Kinzler K.W. Vogelstein B. Analysis of mutations in DNA isolated from plasma and stool of colorectal cancer patients.Gastroenterology. 2008; 135: 489-498Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar Thus, early detection of colon cancer by currently available screening methods remains a major challenge. Circulating DNA has been studied for decades for cancer detection, including detection of CRC.12Diehl F. Schmidt K. Durkee K.H. Moore K.J. Goodman S.N. Shuber A.P. Kinzler K.W. Vogelstein B. Analysis of mutations in DNA isolated from plasma and stool of colorectal cancer patients.Gastroenterology. 2008; 135: 489-498Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 13Anker P. Quantitative aspects of plasma/serum DNA in cancer patients.Ann N Y Acad Sci. 2000; 906: 5-7Crossref PubMed Scopus (34) Google Scholar, 14Anker P. Mulcahy H. Chen X.Q. Stroun M. Detection of circulating tumour DNA in the blood (plasma/serum) of cancer patients.Cancer Metastasis Rev. 1999; 18: 65-73Crossref PubMed Scopus (410) Google Scholar, 15Diehl F. Schmidt K. Choti M.A. Romans K. Goodman S. Li M. Thornton K. Agrawal N. Sokoll L. Szabo S.A. Kinzler K.W. Vogelstein B. Diaz Jr, L.A. Circulating mutant DNA to assess tumor dynamics.Nat Med. 2008; 14: 985-990Crossref PubMed Scopus (2010) Google Scholar, 16Gautschi O. Bigosch C. Huegli B. Jermann M. Marx A. ChassÉ E. Ratschiller D. Weder W. Joerger M. Betticher D.C. Stahel R.A. Ziegler A. Circulating deoxyribonucleic acid as prognostic marker in non-small-cell lung cancer patients undergoing chemotherapy.J Clin Oncol. 2004; 22: 4157-4164Crossref PubMed Scopus (236) Google Scholar, 17Herman J.G. Circulating methylated DNA.Ann N Y Acad Sci. 2004; 1022: 33-39Crossref PubMed Scopus (28) Google Scholar, 18Mulcahy H.E. Lyautey J. Lederrey C. Chen X.Q. Lefort F. Vasioukhin V. Anker P. Alstead E.M. Farthing M.J. Stroun M. Plasma DNA k-ras mutations in patients with gastrointestinal malignancies.Ann N Y Acad Sci. 2000; 906: 25-28Crossref PubMed Scopus (23) Google Scholar, 19Ryan B.M. Lefort F. McManus R. Daly J. Keeling P.W.N. Weir D.G. Kelleher D. A prospective study of circulating mutant kras2 in the serum of patients with colorectal neoplasia: strong prognostic indicator in postoperative follow up.Gut. 2003; 52: 101-108Crossref PubMed Scopus (100) Google Scholar, 20Shirahata A. Sakuraba K. Goto T. Saito M. Ishibashi K. Kigawa G. Nemoto H. Hibi K. Detection of vimentin (vim) methylation in the serum of colorectal cancer patients.Anticancer Res. 2010; 30: 5015-5018PubMed Google Scholar, 21Grutzmann R. Molnar B. Pilarsky C. Habermann J.K. Schlag P.M. Saeger H.D. Miehlke S. Stolz T. Model F. Roblick U.J. Bruch H.P. Koch R. Liebenberg V. Devos T. Song X. Day R.H. Sledziewski A.Z. Lofton-Day C. Sensitive detection of colorectal cancer in peripheral blood by septin 9 DNA methylation assay.PLoS One. 2008; 3: e3759Crossref PubMed Scopus (327) Google Scholar, 22Lofton-Day C. Model F. DeVos T. Tetzner R. Distler J. Schuster M. Song X. Lesche R. Liebenberg V. Ebert M. Molnar B. Grützmann R. Pilarsky C. Sledziewski A. DNA methylation biomarkers for blood-based colorectal cancer screening.Clin Chem. 2008; 54: 414-423Crossref PubMed Scopus (414) Google Scholar However, it has not proved to be sufficiently sensitive to detect tumor-associated DNA alterations in the circulation. We and others have shown that urine contains DNA from the circulation15Diehl F. Schmidt K. Choti M.A. Romans K. Goodman S. Li M. Thornton K. Agrawal N. Sokoll L. Szabo S.A. Kinzler K.W. Vogelstein B. Diaz Jr, L.A. Circulating mutant DNA to assess tumor dynamics.Nat Med. 2008; 14: 985-990Crossref PubMed Scopus (2010) Google Scholar, 23Lichtenstein A.V. Melkonyan H.S. Tomei D. Umansky S.R. Circulating nucleic acids and apoptosis.Ann N Y Acad Sci. 2001; 945: 239-249Crossref PubMed Scopus (137) Google Scholar, 24Su Y.H. Wang M. Brenner D.E. Ng A. Melkonyan H. Umansky S. Syngal S. Block T.M. Human urine contains small, 150 to 250 nucleotide-sized, soluble DNA derived from the circulation and may be useful in the detection of colorectal cancer.J Mol Diagn. 2004; 6: 101-107Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 25Chan A.K.C. Chiu R.W.K. Lo Y.M.D. Cell-free nucleic acids in plasma, serum and urine: a new tool in molecular diagnosis.Ann Clin Biochem. 2003; 40: 122-130Crossref PubMed Scopus (98) Google Scholar, 26Pathak A.K. Bhutani M. Kumar S. Mohan A. Guleria R. Circulating cell-free DNA in plasma/serum of lung cancer patients as a potential screening and prognostic tool.Clin Chem. 2006; 52: 1833-1842PubMed Google Scholar, 27Anker P. Lyautey J. Lederrey C. Stroun M. Circulating nucleic acids in plasma or serum.Clin Chim Acta. 2001; 313: 143-146Crossref PubMed Scopus (79) Google Scholar, 28Jahr S. Hentze H. Englisch S. Hardt D. Fackelmayer F.O. Hesch R. Knippers R. DNA fragments in the blood plasma of cancer patients: quantitations and evidence for their origin from apoptotic and necrotic cells.Cancer Res. 2001; 61: 1659-1665PubMed Google Scholar, 29Stroun M. Maurice P. Vasioukhin V. Lyautey J. Lederrey C. Lefort F. Rossier A. Chen X.Q. Anker P. The origin and mechanism of circulating DNA.Ann N Y Acad Sci. 2000; 906: 161-168Crossref PubMed Scopus (322) Google Scholar and that circulation-derived DNA in urine is fragmented into segments of <300 bp [low-molecular-weight (LMW) urine DNA] that can be used to detect cancer-derived genetic mutations if the tumor is present. We have also shown that preferentially isolating LMW urine DNA from total urine DNA to use as the substrate enhanced the sensitivity and specificity of the test for detecting tumor-derived circulating K-ras–mutated DNA marker.30Su Y.-H. Song J. Wang Z. Wang X. Wang M. Brenner D.E. Block T.M. Removal of high molecular weight DNA by carboxylated magnetic beads enhances the detection of mutated k-ras DNA in urine.Ann N Y Acad Sci. 2008; 1137: 82-91Crossref PubMed Scopus (38) Google Scholar Thus, we suggest that LMW DNA in urine could be used as a substrate for detecting circulation-derived DNA markers. However, because tumor-derived circulating DNA in urine is fragmented,24Su Y.H. Wang M. Brenner D.E. Ng A. Melkonyan H. Umansky S. Syngal S. Block T.M. Human urine contains small, 150 to 250 nucleotide-sized, soluble DNA derived from the circulation and may be useful in the detection of colorectal cancer.J Mol Diagn. 2004; 6: 101-107Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 31Wang M. Block T.M. Steel L. Brenner D.E. Su Y.H. Preferential isolation of fragmented DNA enhances the detection of circulating mutated k-ras DNA.Clin Chem. 2004; 50: 211-213Crossref PubMed Scopus (42) Google Scholar several researchers, including us, have suggested that an assay targeting a small template size is required to have sufficient sensitivity to detect the DNA of interest.12Diehl F. Schmidt K. Durkee K.H. Moore K.J. Goodman S.N. Shuber A.P. Kinzler K.W. Vogelstein B. Analysis of mutations in DNA isolated from plasma and stool of colorectal cancer patients.Gastroenterology. 2008; 135: 489-498Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 32Chan K.C.A. Leung S.F. Yeung S.W. Chan A.T.C. Lo Y.M.D. Quantitative analysis of the transrenal excretion of circulating EBV DNA in nasopharyngeal carcinoma patients.Clin Cancer Res. 2008; 14: 4809-4813Crossref PubMed Scopus (53) Google Scholar, 33Melkonyan H.S. Feaver W.J. Meyer E. Scheinker V. Shekhtman E.M. Xin Z. Umansky S.R. Transrenal nucleic acids: from proof of principle to clinical tests; problems and solutions.Ann N Y Acad Sci. 2008; 1137: 73-81Crossref PubMed Scopus (90) Google Scholar, 34Shekhtman E.M. Anne K. Melkonyan H.S. Robbins D.J. Warsof S.L. Umansky S.R. Optimization of transrenal DNA analysis: detection of fetal DNA in maternal urine.Clin Chem. 2009; 55: 723-729Crossref PubMed Scopus (52) Google Scholar, 35Sikora A. Zimmermann G. Rusterholz C. Birri D. Kolla V. Lapaire O. Hoesli I. Kiefer V. Jackson L. Hahn S. Detection of increased amounts of cell-free fetal DNA with short PCR amplicons.Clin Chem. 2010; 56: 136-138Crossref PubMed Scopus (45) Google Scholar, 36Su Y.-H. Wang M. Norton P.A. Brenner D.E. Block T.M. Detection of mutated k-ras DNA in urine, plasma and serum from patients with colorectal carcinoma or adenomatous polyps.Ann N Y Acad Sci. 2008; 1137: 197-201Crossref PubMed Scopus (86) Google Scholar Aberrant hypermethylation of tumor suppressor genes occurs early and throughout the process of colorectal carcinogenesis.5Ahlquist D.A. Skoletsky J.E. Boynton K. Harrington J.J. Mahoney D.W. Pierceall W.E. Thibodeau S.N. Shuber A.P. Colorectal cancer screening by detection of altered human DNA in stool: feasibility of a multitarget assay panel.Gastroenterology. 2000; 119: 1219-1227Abstract Full Text Full Text PDF PubMed Scopus (503) Google Scholar, 37Ahlquist D.A. Molecular detection of colorectal neoplasia.Gastroenterology. 2010; 138: 2127-2139Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 38Zou H. Harrington J.J. Shire A.M. Rego R.L. Wang L. Campbell M.E. Oberg A.L. Ahlquist D.A. Highly methylated genes in colorectal neoplasia: implications for screening.Cancer Epidemiol Biomarkers Prev. 2007; 16: 2686-2696Crossref PubMed Scopus (103) Google Scholar, 39Itzkowitz S.H. Jandorf L. Brand R. Rabeneck L. Schroy Iii P.C. Sontag S. Johnson D. Skoletsky J. Durkee K. Markowitz S. Shuber A. Improved fecal DNA test for colorectal cancer screening.Clin Gastroenterol Hepatol. 2007; 5: 111-117Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 40Kimura N. Nagasaka T. Murakami J. Sasamoto H. Murakami M. Tanaka N. Matsubara N. Methylation profiles of genes utilizing newly developed CpG island methylation microarray on colorectal cancer patients.Nucleic Acids Res. 2005; 33: e46Crossref PubMed Scopus (28) Google Scholar, 41Grady W.M. Carethers J.M. Genomic and epigenetic instability in colorectal cancer pathogenesis.Gastroenterology. 2008; 135: 1079-1099Abstract Full Text Full Text PDF PubMed Scopus (707) Google Scholar, 42Iacobuzio-Donahue C.A. Epigenetic changes in cancer.Annu Rev Pathol. 2009; 4: 229-249Crossref PubMed Scopus (149) Google Scholar, 43Kim M. Lee J. Sidransky D. DNA methylation markers in colorectal cancer.Cancer Metastasis Rev. 2010; 29: 181-206Crossref PubMed Scopus (244) Google Scholar These DNA alterations could be used as biomarkers for cancer detection and disease management.37Ahlquist D.A. Molecular detection of colorectal neoplasia.Gastroenterology. 2010; 138: 2127-2139Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 43Kim M. Lee J. Sidransky D. DNA methylation markers in colorectal cancer.Cancer Metastasis Rev. 2010; 29: 181-206Crossref PubMed Scopus (244) Google Scholar, 44Markowitz S.D. Bertagnolli M.M. Molecular basis of colorectal cancer.N Engl J Med. 2009; 361: 2449-2460Crossref PubMed Scopus (1456) Google Scholar Among the epigenetic DNA markers, the aberrant hypermethylated vimentin gene (mVIM) has shown great promise in fecal DNA tests for CRC screening8Ahlquist D.A. Sargent D.J. Loprinzi C.L. Levin T.R. Rex D.K. Ahnen D.J. Knigge K. Lance M.P. Burgart L.J. Hamilton S.R. Allison J.E. Lawson M.J. Devens M.E. Harrington J.J. Hillman S.L. Stool DNA and occult blood testing for screen detection of colorectal neoplasia.Ann Intern Med. 2008; 149: 441-450Crossref PubMed Scopus (247) Google Scholar, 37Ahlquist D.A. Molecular detection of colorectal neoplasia.Gastroenterology. 2010; 138: 2127-2139Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 38Zou H. Harrington J.J. Shire A.M. Rego R.L. Wang L. Campbell M.E. Oberg A.L. Ahlquist D.A. Highly methylated genes in colorectal neoplasia: implications for screening.Cancer Epidemiol Biomarkers Prev. 2007; 16: 2686-2696Crossref PubMed Scopus (103) Google Scholar, 39Itzkowitz S.H. Jandorf L. Brand R. Rabeneck L. Schroy Iii P.C. Sontag S. Johnson D. Skoletsky J. Durkee K. Markowitz S. Shuber A. Improved fecal DNA test for colorectal cancer screening.Clin Gastroenterol Hepatol. 2007; 5: 111-117Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 45Chen W.D. Han Z.J. Skoletsky J. Olson J. Sah J. Myeroff L. Platzer P. Lu S. Dawson D. Willis J. Pretlow T.P. Lutterbaugh J. Kasturi L. Willson J.K.V. Rao J.S. Shuber A. Markowitz S.D. Detection in fecal DNA of colon cancer-specific methylation of the nonexpressed vimentin gene.J Natl Cancer Inst. 2005; 97: 1124-1132Crossref PubMed Scopus (330) Google Scholar, 46Zou H. Harrington J. Rego R.L. Ahlquist D.A. A novel method to capture methylated human DNA from stool: implications for colorectal cancer screening.Clin Chem. 2007; 53: 1646-1651Crossref PubMed Scopus (45) Google Scholar, 47Zou H. Taylor W.R. Harrington J.J. Hussain F.T.N. Cao X. Loprinzi C.L. Levine T.R. Rex D.K. Ahnen D. Knigge K.L. Lance P. Jiang X. Smith D.I. Ahlquist D.A. High detection rates of colorectal neoplasia by stool DNA testing with a novel digital melt curve assay.Gastroenterology. 2009; 136: 459-470Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar and was detected in the serum of patients with CRC.20Shirahata A. Sakuraba K. Goto T. Saito M. Ishibashi K. Kigawa G. Nemoto H. Hibi K. Detection of vimentin (vim) methylation in the serum of colorectal cancer patients.Anticancer Res. 2010; 30: 5015-5018PubMed Google Scholar Detection of methylated DNA markers has been challenging when the source of substrate DNA was fragmented or in low quantity, such as DNA isolated from formalin-fixed, paraffin-embedded sections, because the process of bisulfite (BS) conversion further fragments the DNA,48Tanaka K. Okamoto A. Degradation of DNA by bisulfite treatment.Bioorg Med Chem Lett. 2007; 17: 1912-1915Crossref PubMed Scopus (163) Google Scholar, 49Raizis A.M. Schmitt F. Jost J.P. A bisulfite method of 5-methylcytosine mapping that minimizes template degradation.Anal Biochem. 1995; 226: 161-166Crossref PubMed Scopus (168) Google Scholar and it was suggested that approximately 99.9% of the DNA was lost in this process.48Tanaka K. Okamoto A. Degradation of DNA by bisulfite treatment.Bioorg Med Chem Lett. 2007; 17: 1912-1915Crossref PubMed Scopus (163) Google Scholar To detect circulation-derived mVIM in the urine of patients with CRC in this study, we developed an assay targeting a 39-nucleotide (nt) segment of the hypermethylated region in the vimentin gene that was associated with CRC. Using LMW urine DNA as the substrate and the short amplicon assay that we developed, we demonstrated that the circulation-derived, CRC-associated methylated DNA marker mVIM can be detected in the urine of patients with CRC. The potential of this assay for developing a urine test to detect CRC is discussed. Participants were recruited from the Great Lakes–New England Clinical Epidemiology and Validation Center (Ann Arbor, MI) under institutional review board approval. Patients with cancer were enrolled from surgical or oncologic services before treatment, and controls with "no known neoplasia" were enrolled from endoscopy suites, where they had undergone colonoscopies that yielded negative results. All the participants gave informed consent. The clinical profiles of patients with CRC and no-known-neoplasia controls are summarized in Table 1.Table 1Clinical Profiles of Patients with CRC and No-Known-Neoplasia ControlsIDCRCNo known neoplasia⁎Controls with no known neoplasia were enrolled from endoscopy suites, where they had undergone colonoscopies that yielded negative results.Urine sample IDTissue sample IDTNMStageAge (years)SexUrine sample IDAge (years)SexAU20T152/0/?I63MN175FBU19T132/1/?IIIa69MN263FCU12T143/1/?58MN371FDU11T173/1/XIIIb79FN478MEU1T53/1/1IV54MN572MFU5T123/0/XIIa75FN679MGU18T181/0/0I80MN779FHU3T84/0/1IV45MN885MIU14T203/2/XIIIc69MN982FJU16T102/0/XIc69MN1068FKU8T93/0/XIIa36MN1166MLU10T23/2/1IV64MN1269FMU9T163/0/XIIa65FN1377FNU15T44/0/0IIb32MN1465FOU17T64/2/1IV59FN1567FPU13T193/0/XIIa74MN1682MQU7T111/0/XI46FN1769FRU4T33/0/0IIa46MN1888MSU6T73/2/0IIIc67FN1962MTU2T13/0/XIIa59MN2079FAge, mean (years) (P = 0.00048†By Student's t-test.)60.4573.8Sex, F/M (No.) (P = 0.11‡By Fisher's exact test.)6/1412/8F, female; M, male; TNM, tumor node metastasis. Controls with no known neoplasia were enrolled from endoscopy suites, where they had undergone colonoscopies that yielded negative results.† By Student's t-test.‡ By Fisher's exact test. Open table in a new tab F, female; M, male; TNM, tumor node metastasis. A total of 0.5 mol/L EDTA, pH 8.0, was added to freshly collected urine to a final concentration of 10 mmol/L EDTA to inhibit possible nuclease activity; the mixture was stored at −70°C. To isolate total urine DNA, the frozen urine sample was thawed at room temperature and then placed immediately in ice before the DNA was isolated. Total urine DNA was isolated from the thawed urine within an hour as described previously.24Su Y.H. Wang M. Brenner D.E. Ng A. Melkonyan H. Umansky S. Syngal S. Block T.M. Human urine contains small, 150 to 250 nucleotide-sized, soluble DNA derived from the circulation and may be useful in the detection of colorectal cancer.J Mol Diagn. 2004; 6: 101-107Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar The LMW urine DNA and high-molecular-weight (HMW) urine DNA fractions were obtained using carboxylated magnetic beads (Agencourt Bioscience Corp., Beverly, MA) and a binding method developed previously by our laboratory (Philadelphia, PA).30Su Y.-H. Song J. Wang Z. Wang X. Wang M. Brenner D.E. Block T.M. Removal of high molecular weight DNA by carboxylated magnetic beads enhances the detection of mutated k-ras DNA in urine.Ann N Y Acad Sci. 2008; 1137: 82-91Crossref PubMed Scopus (38) Google Scholar DNA from paraffin-embedded tissue sections was isolated using the MasterPure DNA kit (Epicentre Biotechnologies, Madison, WI) per the manufacturer's instructions. BS conversion of DNA was performed using EpiTect BS conversion kits (Qiagen Inc, Valencia, CA) according to the manufacturer's specifications. To evaluate the efficiency of the BS conversion, BS-converted DNA was subjected to PCR amplification using BS-specific primer sets, as described previously.14Anker P. Mulcahy H. Chen X.Q. Stroun M. Detection of circulating tumour DNA in the blood (plasma/serum) of cancer patients.Cancer Metastasis Rev. 1999; 18: 65-73Crossref PubMed Scopus (410) Google Scholar The PCR product was analyzed and visualized by gel containing ethidium bromide, excised, and purified by the Qiagen gel extraction kit (Qiagen Inc.); it was then sent to NapCore (Children's Hospital of Philadelphia, Philadelphia, PA) for DNA sequencing. The DNA sequencing data were compared with the sequence generated by the Methyl Primer Express software (Applied Biosystems, Foster City, CA) using ClustalW software (European Bioinformatics Institute, Cambridge, UK). The efficiency of the BS conversion was determined as the percentage of the number of non-CpG cytosine molecules that became thymidine compared with the percentage of the total number of non-CpG cytosine molecules in the region. Only samples that were converted with an efficiency of ≥95% were analyzed. DNA was quantified by a real-time PCR amplifying globin DNA as previously described.50Lin S.Y. Dhillon V. Jain S. Chang T.T. Hu C.T. Lin Y.J. Chen S.H. Chang K.C. Song W. Yu L. Block T.M. Su Y.H. A locked nucleic acid clamp-mediated PCR assay for detection of a p53 codon 249 hotspot mutation in urine.J Mol Diagn. 2011; 13: 474-484Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar To quantify the BS-converted DNA, we developed a real-time PCR assay, BS-actin, targeting the BS-converted actin gene sequences. The primers, listed in Table 2, were designed within the regions that did not have any CpG sites in the gene so that the status of CpG methylation would not affect the primer binding. The BS-actin PCR was performed using the LightCycler 480 real-time PCR system (Roche Biochemical, Mannheim, Germany) and the LightCycler 480 SYBR Green I master kit (Roche Biochemical). The reaction contained 1× SYBR Green master mix, 1.0 μmol/L primers, and the BS-converted DNA template. The PCR was performed under the following conditions: 95°C for 10 minutes to activate the Taq polymerase, then 95°C for 10 seconds, 55°C for 20 seconds, 72°C for 10 seconds for 45 cycles, followed by determination of the melting curve at 95°C for 5 seconds, 65°C for 1 minute, and 97°C for continuous hold. Cooling occurred at 40°C for 30 seconds. The linearity and sensitivity of the assay were determined by performing the assay using a 10-fold dilution of the BS-converted human universal methylated DNA control (Zymo Research, Irvine, CA) (see Supplemental Figure S1 at http://jmd.amjpathol.org).Table 2Oligonucleotides Used in the StudyAssay (Genbank)Oligonucleotide sequences⁎The artificial DNA sequences are underlined, and LNAs are in bold.Annealing temperature (°C)BS-actin (NT_007819)Forward: 5′-GATGTATGAAGGTTTTTGG-3′55Reverse: 5′-CTAACTACCTCCACCCACTC-3′MSP2945Chen W.D. Han Z.J. Skoletsky J. Olson J. Sah J. Myeroff L. Platzer P. Lu S. Dawson D. Willis J. Pretlow T.P. Lutterbaugh J. Kasturi L. Willson J.K.V. Rao J.S. Shuber A. Markowitz S.D. Detection in fecal DNA of colon cancer-specific methylation of the nonexpressed vimentin gene.J Natl Cancer Inst. 2005; 97: 1124-1132Crossref PubMed Scopus (330) Google Scholar (AL133415)MSP29F: 5′-TCGTTTCGAGGTTTTCGCGTTAGAGAC-3′68MSP29R: 5′-CGACTAAAACTCGACCGACTCGCGA-3′VIM29R_LNA 2-step MethyLight1F: 5′-GCTCTTCGTGGTGTGGTGCGGTTCGGGTATCGC-3′First PCR: 621R: 5′-GCTCTTCGTGGTGTGGTGCTCCGACTAAAACTCGACC-3′2F: 5′-GTGTGGTGCGGTTC-3′Second PCR: 602R: 5′-GTGTGGTGCTCCGAC-3′TaqMan probe: FAM-ATCGCGAGTCGGTCGAGTT-BHQ1 The artificial DNA sequences are underlined, and LNAs are in bold. Open table in a new tab To develop the MethyLight assay for mVIM, target sequences from the promoter and first exon regions of the vimentin gene were obtained from GenBank (AL133415) using PubMed software (National Center for Biotechnology Information, Bethesda, MD). CpG analysis was performed using Methyl Primer Express software (Applied Biosystems). Primers and probes for a two-step nested MethyLight assay targeting template sizes of 39 nt were designed (Table 2). The first PCR was performed in a thermocycler (Eppendorf, Hamburg, Germany) using the first PCR primer set (0.1 μmol/L), dNTP (20 μmol/L), and HotStar Taq (Qiagen Inc.) under the following conditions: 95°C for 15 minutes to activate the HotStar Taq polymerase, then 95°C for 30 seconds, 62°C for 30 seconds, 72°C for 20 seconds for 30 cycles, and 72°C for 5 minutes. The second PCR was perfo
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