title: Cancer detection by ubiquitin carboxyl-terminal esterase L1 methylation in pancreatobiliary fluids.
abstract: OBJECTIVE
  To evaluate the utility of measuring epigenetic alterations in pancreatic and biliary fluids in determining molecular markers for pancreatobiliary cancers.
  
  
  METHODS
  DNA was extracted from undiluted pancreatic and biliary fluids. As a surrogate for a genome-wide hypomethylation assay, levels of long interspersed nuclear element-1 (LINE-1) methylation were analyzed using bisulfite pyrosequencing. CpG island hypermethylation of 10 tumor-associated genes, aryl-hydrocarbon receptor repressor, adenomatous polyposis coli, calcium channel, voltage dependent, T type α1G subunit, insulin-like growth factor 2, O-6-methyl-guanine-DNA methyltransferase, neurogenin 1, CDKN2A, runt-related transcription factor 3 (RUNX3), secreted frizzled-related protein 1, and ubiquitin carboxyl-terminal esterase L1 (UCHL1), was analyzed using MethyLight. To examine the role of CpG methylation and histone deacetylation in the silencing of UCHL1, human gallbladder carcinoma cell lines and pancreatic carcinoma cell lines were treated with 2 or 5 μmol/L 5-AZA-dC for 72 h or 100 nmol/L Trichostatin A for 24 h. After the treatment, UCHL1 expression was analyzed by real-time reverse transcription-polymerase chain reaction.
  
  
  RESULTS
  Pancreatobiliary cancers exhibited significantly lower LINE-1 methylation levels in pancreatic and biliary fluids than did noncancerous pancreatobiliary disease (58.7% ± 4.3% vs 61.7% ± 2.2%, P = 0.027; 53.8% ± 6.6% vs 57.5% ± 1.7%, P = 0.007); however, LINE-1 hypomethylation was more evident in pancreatic cancer tissues than in pancreatic fluids (45.4% ± 5.5% vs 58.7% ± 4.3%, P < 0.001). CpG island hypermethylation of tumor-associated genes was detected at various frequencies, but it was not correlated with LINE-1 hypomethylation. Hypermethylation of the UCHL1 gene was cancer-specific and most frequently detected in pancreatic (67%) or biliary (70%) fluids from patients with pancreatobiliary cancer. As a single marker, hypermethylation of the UCHL1 gene in pancreatic and biliary fluids was most useful for the detection of pancreatic and pancreatobiliary cancers, respectively (100% specificity). Hypermethylation of the UCHL1 and RUNX3 genes in pancreatic and biliary fluids was the most useful combined marker for pancreatic (87% sensitivity and 100% specificity) and pancreatobiliary (97% sensitivity and 100% specificity) cancers. Treatment with a demethylating agent, 5-AZA-2'-deoxycytidine, restored UCHL1 expression in pancreatobiliary cancer cell lines.
  
  
  CONCLUSIONS
  Our results suggest that hypermethylation of UCHL1 and RUNX3 in pancreatobiliary fluid might be useful for the diagnosis of pancreatobiliary cancers.
authors.affiliation: First Department of Internal Medicine, Sapporo Medical University School of Medicine, Sapporo 060-8543, Japan.
authors.forename: Norihiro
authors.initials: N
authors.lastname: Kato
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Yamamoto
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Adachi
authors.forename: Hirokazu
authors.initials: H
authors.lastname: Ohashi
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Taniguchi
authors.forename: Hiromu
authors.initials: H
authors.lastname: Suzuki
authors.forename: Mayumi
authors.initials: M
authors.lastname: Nakazawa
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Kaneto
authors.forename: Shigeru
authors.initials: S
authors.lastname: Sasaki
authors.forename: Kohzoh
authors.initials: K
authors.lastname: Imai
authors.forename: Yasuhisa
authors.initials: Y
authors.lastname: Shinomura
mesh_terms: D000107:Acetylation; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000704:Analysis of Variance; D000818:Animals; D001646:Bile / Q000201:enzymology; D001661:Biliary Tract Neoplasms / Q000201:enzymology* / Q000235:genetics; D014408:Biomarkers, Tumor / Q000235:genetics / Q000378:metabolism*; D002415:Cats; D045744:Cell Line, Tumor; D016009:Chi-Square Distribution; D050677:Core Binding Factor Alpha 3 Subunit / Q000235:genetics / Q000378:metabolism; D018899:CpG Islands; D019175:DNA Methylation; D015254:DNA Modification Methylases / Q000037:antagonists & inhibitors / Q000378:metabolism; D005260:Female; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology; D006657:Histones / Q000378:metabolism; D006801:Humans; D020084:Long Interspersed Nucleotide Elements; D008297:Male; D008875:Middle Aged; D010189:Pancreatic Juice / Q000201:enzymology; D010190:Pancreatic Neoplasms / Q000201:enzymology* / Q000235:genetics; D043222:Ubiquitin Thiolesterase / Q000235:genetics / Q000378:metabolism*
chemical_list: D014408:Biomarkers, Tumor; D050677:Core Binding Factor Alpha 3 Subunit; D056572:Histone Deacetylase Inhibitors; D006657:Histones; C493729:Runx3 protein, human; C482860:UCHL1 protein, human; D015254:DNA Modification Methylases; D043222:Ubiquitin Thiolesterase
keywords: DNA methylation; Pancreatobiliary cancers; Pancreatobiliary fluids; Runt-related transcription factor 3; Ubiquitin carboxyl-terminal esterase L1
references.citation: Nucleic Acids Res. 2004 Feb 18;32(3):e38
references.pmid: 14973332
references.citation: Cancer Biol Ther. 2006 Oct;5(10):1392-9
references.pmid: 17106238
references.citation: Cancer Res. 2000 Apr 1;60(7):1835-9
references.pmid: 10766168
references.citation: Clin Chim Acta. 2007 Apr;379(1-2):127-33
references.pmid: 17303099
references.citation: J Hepatobiliary Pancreat Surg. 2006;13(4):286-95
references.pmid: 16858539
references.citation: Virchows Arch. 2009 Oct;455(4):343-51
references.pmid: 19763613
references.citation: Cancer Epidemiol Biomarkers Prev. 2008 Oct;17(10):2555-64
references.pmid: 18842996
references.citation: Am J Gastroenterol. 2004 Sep;99(9):1675-81
references.pmid: 15330900
references.citation: Science. 2003 Apr 18;300(5618):455
references.pmid: 12702868
references.citation: Cancer Res. 2006 Aug 15;66(16):7920-8
references.pmid: 16912165
references.citation: Cancer Res. 2003 May 15;63(10):2649-57
references.pmid: 12750293
references.citation: Int J Cancer. 2008 Aug 15;123(4):753-9
references.pmid: 18512240
references.citation: Nat Rev Genet. 2002 Jun;3(6):415-28
references.pmid: 12042769
references.citation: Mod Pathol. 2007 Jul;20(7):802-10
references.pmid: 17483816
references.citation: Cancer Res. 2006 Jan 15;66(2):1208-17
references.pmid: 16424060
references.citation: Nat Rev Cancer. 2011 Jun;11(6):426-37
references.pmid: 21562580
references.citation: J Mol Diagn. 2006 May;8(2):209-17
references.pmid: 16645207
references.citation: Clin Cancer Res. 2005 Mar 15;11(6):2285-92
references.pmid: 15788678
references.citation: Cancer Res. 2003 Jul 1;63(13):3735-42
references.pmid: 12839967
references.citation: Clin Gastroenterol Hepatol. 2008 Nov;6(11):1270-8
references.pmid: 18995218
references.citation: Curr Probl Surg. 2007 Jul;44(7):396-482
references.pmid: 17693325
references.citation: Nat Rev Genet. 2009 Oct;10(10):691-703
references.pmid: 19763152
references.citation: Int J Cancer. 2008 Jun 15;122(12):2767-73
references.pmid: 18366060
references.citation: Carcinogenesis. 2007 Jun;28(6):1364-70
references.pmid: 17183069
references.citation: Nat Rev Cancer. 2010 Oct;10(10):683-95
references.pmid: 20814421
references.citation: Nucleic Acids Res. 2005 Dec 02;33(21):6823-36
references.pmid: 16326863
references.citation: Oncogene. 2004 Nov 18;23(54):8841-6
references.pmid: 15480421
references.citation: World J Gastroenterol. 2008 Oct 7;14(37):5650-6
references.pmid: 18837080
references.citation: Gastroenterology. 2003 May;124(5):1300-10
references.pmid: 12730870
references.citation: Oncogene. 2005 Nov 24;24(53):7946-52
references.pmid: 16007117
references.citation: Oncogene. 2004 Mar 25;23(13):2401-7
references.pmid: 14743205
references.citation: Cancer Res. 2004 Feb 1;64(3):871-5
references.pmid: 14871814
references.citation: Am J Clin Pathol. 2007 Aug;128(2):272-9
references.pmid: 17638662
references.citation: J Clin Oncol. 2010 Jul 20;28(21):3531-40
references.pmid: 20547994
references.citation: J Biosci. 2012 Jun;37(2):301-12
references.pmid: 22581336
references.citation: Br J Cancer. 2008 May 20;98(10):1690-5
references.pmid: 18475302
references.citation: Cancer Biol Ther. 2003 Jan-Feb;2(1):78-83
references.pmid: 12673124
references.citation: Nat Genet. 2006 Jul;38(7):787-93
references.pmid: 16804544
references.citation: Clin Cancer Res. 2010 Nov 1;16(21):5114-23
references.pmid: 20978145
references.citation: Cancer Res. 2003 Jul 15;63(14):4158-66
references.pmid: 12874021
references.citation: Crit Rev Oncol Hematol. 2007 Jan;61(1):44-51
references.pmid: 17164111