title: Breast cancer metastasis suppressor-1 promoter methylation in primary breast tumors and corresponding circulating tumor cells.
abstract: Breast cancer metastasis suppressor-1 (BRMS1) differentially regulates the expression of multiple genes, leading to metastasis suppression without affecting orthotopic tumor growth. For the first time, BRMS1 promoter methylation was evaluated as a prognostic biomarker in primary breast tumors and a subset of corresponding circulating tumor cells (CTC). Formalin-fixed paraffin embedded samples were analyzed for BRMS1 methylation status using methylation-specific PCR in a human specimen cohort consisting of noncancerous tissues, benign fibroadenomas, and primary breast tumors, including some with adjacent noncancerous tissues. Peripheral blood mononuclear cells from a large subset of these patients were fixed in cytospins and analyzed. In addition, BRMS1 expression in cytospins was examined by double-immunofluorescence using anti-BRMS1 and pan-cytokeratin antibodies. BRMS1 promoter methylation was not detected in noncancerous breast tissues or benign fibroadenomas; however, methylation was observed in more than a third of primary breast tumors. Critically, BRMS1 promoter methylation in primary tumors was significantly associated with reduced disease-free survival with a trend toward reduced overall survival. Similarly, a third of cytospin samples were positive for the presence of CTCs, and the total number of detected CTCs was 41. Although a large fraction of CTCs were negative or maintained low expression of BRSM1, promoter methylation was observed in a small fraction of samples, implying that BRSM1 expression in CTCs was either downregulated or heterogeneous. In summary, these data define BRMS1 promoter methylation in primary breast tumors and associated CTCs.
  
  
  CONCLUSIONS
  This study indicates that BRSM1 promoter methylation status has biomarker potential in breast cancer.
authors.affiliation: Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, Athens 15771, Greece. lianidou@chem.uoa.gr.
authors.forename: Maria
authors.initials: M
authors.lastname: Chimonidou
authors.forename: Galatea
authors.initials: G
authors.lastname: Kallergi
authors.forename: Vassilis
authors.initials: V
authors.lastname: Georgoulias
authors.forename: Danny R
authors.initials: DR
authors.lastname: Welch
authors.forename: Evi S
authors.initials: ES
authors.lastname: Lianidou
mesh_terms: D000328:Adult; D014408:Biomarkers, Tumor / Q000235:genetics; D001943:Breast Neoplasms / Q000175:diagnosis / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D019175:DNA Methylation; D018572:Disease-Free Survival; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008875:Middle Aged; D009363:Neoplasm Proteins / Q000235:genetics* / Q000378:metabolism; D009364:Neoplasm Recurrence, Local; D009360:Neoplastic Cells, Circulating / Q000378:metabolism* / Q000473:pathology; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins
chemical_list: C410024:BRMS1 protein, human; D014408:Biomarkers, Tumor; D009363:Neoplasm Proteins; D012097:Repressor Proteins