title: Promoter methylation of ITF2, but not APC, is associated with microsatellite instability in two populations of colorectal cancer patients.
abstract: BACKGROUND
  Aberrant Wnt signaling activation occurs commonly in colorectal carcinogenesis, leading to upregulation of many target genes. APC (adenomatous polyposis coli) is an important component of the β-catenin destruction complex, which regulates Wnt signaling, and is often mutated in colorectal cancer (CRC). In addition to mutational events, epigenetic changes arise frequently in CRC, specifically, promoter hypermethylation which silences tumor suppressor genes. APC and the Wnt signaling target gene ITF2 (immunoglobulin transcription factor 2) incur hypermethylation in various cancers, however, methylation-dependent regulation of these genes in CRC has not been studied in large, well-characterized patient cohorts. The microsatellite instability (MSI) subtype of CRC, featuring DNA mismatch repair deficiency and often promoter hypermethylation of MutL homolog 1 (MLH1), has a favorable outcome and is characterized by different chemotherapeutic responses than microsatellite stable (MSS) tumors. Other epigenetic events distinguishing these subtypes have not yet been fully elucidated.
  
  
  METHODS
  Here, we quantify promoter methylation of ITF2 and APC by MethyLight in two case-case studies nested in population-based CRC cohorts from the Ontario Familial Colorectal Cancer Registry (n = 330) and the Newfoundland Familial Colorectal Cancer Registry (n = 102) comparing MSI status groups.
  
  
  RESULTS
  ITF2 and APC methylation are significantly associated with tumor versus normal state (both P < 1.0 × 10(-6)). ITF2 is methylated in 45.8% of MSI cases and 26.9% of MSS cases and is significantly associated with MSI in Ontario (P = 0.002) and Newfoundland (P = 0.005) as well as the MSI-associated feature of MLH1 promoter hypermethylation (P = 6.72 × 10(-4)). APC methylation, although tumor-specific, does not show a significant association with tumor subtype, age, gender, or stage, indicating it is a general tumor-specific CRC biomarker.
  
  
  CONCLUSIONS
  This study demonstrates, for the first time, MSI-associated ITF2 methylation, and further reveals the subtype-specific epigenetic events modulating Wnt signaling in CRC.
authors.affiliation: Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada. savio@lunenfeld.ca.|Lunenfeld-Tanenbaum Research Institute of Mount Sinai Hospital, Toronto, ON, Canada. savio@lunenfeld.ca.
authors.forename: Andrea J
authors.initials: AJ
authors.lastname: Savio
authors.affiliation: Lunenfeld-Tanenbaum Research Institute of Mount Sinai Hospital, Toronto, ON, Canada. daftary@lunenfeld.ca.|Ontario Familial Colorectal Cancer Registry, Toronto, ON, Canada. daftary@lunenfeld.ca.
authors.forename: Darshana
authors.initials: D
authors.lastname: Daftary
authors.affiliation: Faculty of Medicine, Memorial University of Newfoundland, St John's, Newfoundland, Canada. edicks@mun.ca.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Dicks
authors.affiliation: Oncogenomics Group, Genetic Epidemiology Laboratory, Department of Pathology, The University of Melbourne, Parkville, VIC, Australia. daniel.buchanan@unimelb.edu.au.|Centre for Epidemiology and Biostatistics, Melbourne School of Population and Global Health, The University of Melbourne, Parkville, VIC, Australia. daniel.buchanan@unimelb.edu.au.
authors.forename: Daniel D
authors.initials: DD
authors.lastname: Buchanan
authors.affiliation: Faculty of Medicine, Memorial University of Newfoundland, St John's, Newfoundland, Canada. pparfrey@mun.ca.
authors.forename: Patrick S
authors.initials: PS
authors.lastname: Parfrey
authors.affiliation: Department of Haematology and Oncology, The Queen Elizabeth Hospital, Woodville, South Australia, Australia. joanne.young@adelaide.edu.au.
authors.forename: Joanne P
authors.initials: JP
authors.lastname: Young
authors.affiliation: USC Epigenome Center, University of Southern California, Los Angeles, CA, USA. dan.weisenberger@med.usc.edu.
authors.forename: Daniel
authors.initials: D
authors.lastname: Weisenberger
authors.affiliation: Faculty of Medicine, Memorial University of Newfoundland, St John's, Newfoundland, Canada. rcgreen@mun.ca.
authors.forename: Roger C
authors.initials: RC
authors.lastname: Green
authors.affiliation: Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada. steven.gallinger@uhn.ca.|Lunenfeld-Tanenbaum Research Institute of Mount Sinai Hospital, Toronto, ON, Canada. steven.gallinger@uhn.ca.|Ontario Familial Colorectal Cancer Registry, Toronto, ON, Canada. steven.gallinger@uhn.ca.|Department of Surgery, University of Toronto, Toronto, ON, Canada. steven.gallinger@uhn.ca.
authors.forename: Steven
authors.initials: S
authors.lastname: Gallinger
authors.affiliation: Lunenfeld-Tanenbaum Research Institute of Mount Sinai Hospital, Toronto, ON, Canada. john.mclaughlin@oahpp.ca.|Ontario Familial Colorectal Cancer Registry, Toronto, ON, Canada. john.mclaughlin@oahpp.ca.|Dalla Lana School of Public Health, University of Toronto, Toronto, ON, Canada. john.mclaughlin@oahpp.ca.
authors.forename: John R
authors.initials: JR
authors.lastname: McLaughlin
authors.affiliation: Lunenfeld-Tanenbaum Research Institute of Mount Sinai Hospital, Toronto, ON, Canada. knight@lunenfeld.ca.|Dalla Lana School of Public Health, University of Toronto, Toronto, ON, Canada. knight@lunenfeld.ca.
authors.forename: Julia A
authors.initials: JA
authors.lastname: Knight
authors.affiliation: Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada. bapat@lunenfeld.ca.|Lunenfeld-Tanenbaum Research Institute of Mount Sinai Hospital, Toronto, ON, Canada. bapat@lunenfeld.ca.|Department of Pathology, University Health Network, Toronto, ON, Canada. bapat@lunenfeld.ca.
authors.forename: Bharati
authors.initials: B
authors.lastname: Bapat
mesh_terms: D025601:Adenomatous Polyposis Coli Protein / Q000235:genetics*; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors / Q000235:genetics*; D015331:Cohort Studies; D003106:Colon / Q000737:chemistry; D015179:Colorectal Neoplasms / Q000737:chemistry / Q000235:genetics*; D019175:DNA Methylation / Q000235:genetics*; D005260:Female; D006801:Humans; D008297:Male; D053842:Microsatellite Instability; D008875:Middle Aged; D011401:Promoter Regions, Genetic; D000073940:Transcription Factor 4; D014157:Transcription Factors / Q000235:genetics*; D060449:Wnt Signaling Pathway
chemical_list: C555337:APC protein, human; D025601:Adenomatous Polyposis Coli Protein; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; C532096:TCF4 protein, human; D000073940:Transcription Factor 4; D014157:Transcription Factors