title: Mapping geographic zones of cancer risk with epigenetic biomarkers in normal breast tissue.
abstract: OBJECTIVE
  Genetic alterations were previously identified in normal epithelia adjacent to invasive cancers. The aim of this study was to determine DNA methylation in histologically normal tissues from multiple geographic zones adjacent to primary breast tumors.
  
  
  METHODS
  First, methylation status of a 4-kb region of RASSF1A promoter was interrogated using oligonucleotide-based microarray in 144 samples (primary tumors, 47; adjacent normals, 69; reduction mammoplasty tissues, 28). Second, allelic imbalance (AI)/loss of heterozygosity (LOH) surrounding RASSF1A promoter were analyzed in 30 samples (tumors, 8; adjacent normals, 22). Third, global methylation screening of 49 samples (tumors, 12; adjacent normals, 25; reduction mammoplasty, 12) was done by differential methylation hybridization. Real-time quantitative methylation-specific PCR was used to validate the microarray findings.
  
  
  RESULTS
  DNA methylation in the core RASSF1A promoter was low in reduction mammoplasty tissues (P=0.0001) when compared with primary tumors. The adjacent normals had an intermediate level of methylation. The regions surrounding the core were highly methylated in all sample types. Microsatellite markers showed AI/LOH in tumors and some of the adjacent normals. Concurrent AI/LOH and DNA methylation in RASSF1A promoter occurred in two of six tumors. Global methylation screening uncovered genes more methylated in adjacent normals than in reduction mammoplasty tissues. The methylation status of four genes was confirmed by quantitative methylation-specific PCR.
  
  
  CONCLUSIONS
  Our findings suggest a field of methylation changes extending as far as 4 cm from primary tumors. These frequent alterations may explain why normal tissues are at risk for local recurrence and are useful in disease prognostication.
authors.affiliation: Department of Hematology and Oncology, Comprehensive Cancer Center, College of Medicine and Public Health, The Ohio State University, Columbus, Ohio 43210, USA, and Department of Pathology, Addenbrooks' Hospital, Cambridge, UK.
authors.forename: Pearlly S
authors.initials: PS
authors.lastname: Yan
authors.forename: Chinnambally
authors.initials: C
authors.lastname: Venkataramu
authors.forename: Ashraf
authors.initials: A
authors.lastname: Ibrahim
authors.forename: Joseph C
authors.initials: JC
authors.lastname: Liu
authors.forename: Rulong Z
authors.initials: RZ
authors.lastname: Shen
authors.forename: Nils M
authors.initials: NM
authors.lastname: Diaz
authors.forename: Barbara
authors.initials: B
authors.lastname: Centeno
authors.forename: Frank
authors.initials: F
authors.lastname: Weber
authors.forename: Yu-Wei
authors.initials: YW
authors.lastname: Leu
authors.forename: Charles L
authors.initials: CL
authors.lastname: Shapiro
authors.forename: Charis
authors.initials: C
authors.lastname: Eng
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Yeatman
authors.forename: Tim H-M
authors.initials: TH
authors.lastname: Huang
mesh_terms: D022981:Allelic Imbalance; D014408:Biomarkers, Tumor / Q000032:analysis; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology / Q000601:surgery; D002277:Carcinoma / Q000235:genetics* / Q000378:metabolism / Q000473:pathology / Q000601:surgery; D002893:Chromosomes, Human, Pair 3; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D020869:Gene Expression Profiling; D006801:Humans; D019656:Loss of Heterozygosity; D042361:Mammary Glands, Human / Q000378:metabolism*; D008954:Models, Biological; D009364:Neoplasm Recurrence, Local / Q000175:diagnosis; D011379:Prognosis; D011401:Promoter Regions, Genetic; D025521:Tumor Suppressor Proteins / Q000235:genetics*
chemical_list: D014408:Biomarkers, Tumor; C411762:RASSF1 protein, human; D025521:Tumor Suppressor Proteins