title: Applicability of HIN-1, MGMT and RASSF1A promoter methylation as biomarkers for detecting field cancerization in breast cancer.
abstract: BACKGROUND
  It has been shown in some articles that genetic and epigenetic abnormalities cannot only be found in tumor tissues but also in adjacent regions that appear histologically normal. This phenomenon is metaphorically called field cancerization or field defect. Field cancerization is regarded as clinically significant because it is assumed to be an important factor in local recurrence of cancer. As the field showing these molecular abnormalities may not be removed completely by surgery, these changes might lead to neoplasms and subsequent transformation to a tumor. We aimed to investigate the applicability of the methylation status of six tumor suppressor genes as biomarkers for detecting field cancerization in breast cancer.
  
  
  METHODS
  The promoter methylation status of CCND2, DAPK1, GSTP1, HIN-1, MGMT and RASSF1A was determined by methylation-sensitive high-resolution melting (MS-HRM) analysis. MS-HRM methods for CCND2, MGMT and RASSF1A were developed in-house, primer sequences for DAPK1, GSTP1 and HIN-1 have already been published. Biopsy samples were taken from tumor, tumor-adjacent and tumor-distant tissue from 17 breast cancer patients. Normal breast tissues of four healthy women served as controls.
  
  
  RESULTS
  All MS-HRM methods proved to be very sensitive. LODs were in the range from 0.1 to 1.5 %, LOQs ranged from 0.3 to 5.3 %. A total of 94 %, 82 % and 65 % of the tumors showed methylation of RASSF1A, HIN-1 and MGMT promoters, respectively. The methylation status of these promoters was significantly lower in tumor-distant tissues than in tumor tissues. Tumor-adjacent tissues showed higher methylation status of RASSF1A, HIN-1 and MGMT promoters than tumor-distant tissues, indicating field cancerization. The methylation status of the HIN-1 promoter in tumor-adjacent tissues was found to correlate strongly with that in the corresponding tumors (r = 0.785, p < 0.001), but not with that in the corresponding tumor-distant tissues (r = 0.312, p = 0.239).
  
  
  CONCLUSIONS
  Among the gene promoters investigated, the methylation status of the HIN-1 promoter can be considered the best suitable biomarker for detecting field cancerization. Further investigation is needed to test whether it can be used for defining surgical margins in order to prevent future recurrence of breast cancer.
authors.affiliation: Department of Analytical Chemistry, University of Vienna, Währinger Str. 38, 1090, Vienna, Austria. melanie.spitzwieser@univie.ac.at.
authors.forename: Melanie
authors.initials: M
authors.lastname: Spitzwieser
authors.affiliation: Department of Analytical Chemistry, University of Vienna, Währinger Str. 38, 1090, Vienna, Austria. elisabeth_holzweber@gmx.at.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Holzweber
authors.affiliation: Department of Obstetrics and Gynecology, Division of Gynecology and Gynecological Oncology, Medical University of Vienna, Währinger Gürtel 18-20, 1090, Vienna, Austria. georg.pfeiler@meduniwien.ac.at.
authors.forename: Georg
authors.initials: G
authors.lastname: Pfeiler
authors.affiliation: Department of Plastic and Reconstructive Surgery, Medical University of Vienna, Währinger Gürtel 18-20, 1090, Vienna, Austria. stefan.hacker@meduniwien.ac.at.
authors.forename: Stefan
authors.initials: S
authors.lastname: Hacker
authors.affiliation: Department of Analytical Chemistry, University of Vienna, Währinger Str. 38, 1090, Vienna, Austria. margit.cichna@univie.ac.at.
authors.forename: Margit
authors.initials: M
authors.lastname: Cichna-Markl
mesh_terms: D000328:Adult; D000368:Aged; D014408:Biomarkers, Tumor / Q000235:genetics*; D001706:Biopsy; D001943:Breast Neoplasms / Q000235:genetics* / Q000473:pathology; D056742:Cyclin D2 / Q000235:genetics; D016207:Cytokines / Q000235:genetics*; D019175:DNA Methylation / Q000235:genetics*; D015254:DNA Modification Methylases / Q000235:genetics*; D045643:DNA Repair Enzymes / Q000235:genetics*; D064550:Death-Associated Protein Kinases / Q000235:genetics; D044127:Epigenesis, Genetic / Q000235:genetics; D005260:Female; D051549:Glutathione S-Transferase pi / Q000235:genetics; D006801:Humans; D008875:Middle Aged; D009364:Neoplasm Recurrence, Local / Q000235:genetics / Q000473:pathology; D011401:Promoter Regions, Genetic / Q000235:genetics*; D025521:Tumor Suppressor Proteins / Q000235:genetics*
chemical_list: D014408:Biomarkers, Tumor; C080336:CCND2 protein, human; D056742:Cyclin D2; D016207:Cytokines; C411762:RASSF1 protein, human; C434732:SCGB3A1 protein, human; D025521:Tumor Suppressor Proteins; D015254:DNA Modification Methylases; C515491:MGMT protein, human; C496556:GSTP1 protein, human; D051549:Glutathione S-Transferase pi; C579096:DAPK1 protein, human; D064550:Death-Associated Protein Kinases; D045643:DNA Repair Enzymes