title: CST6 promoter methylation in circulating cell-free DNA of breast cancer patients.
abstract: OBJECTIVE
  We have recently shown that detection of CST6 promoter methylation in primary breast tumors can provide important prognostic information in patients with operable breast cancer and that CST6 promoter is also methylated in Circulating Tumor Cells (CTC). In this study we evaluated the presence of CST6 promoter methylation in cell-free DNA (cfDNA) circulating in plasma of breast cancer patients.
  
  
  METHODS
  Our study material consisted of: a) a pilot testing group of 27 patients with stage I-III operable breast cancer, 46 patients with verified metastasis and 37 healthy donors and b) an independent cohort of 123 consecutive stage I-III operable breast cancer patients. Methylated and unmethylated CST6 promoter sequences were detected by using methylation-specific PCR (MSP). CST6 immunohistochemical detection was performed in 20 corresponding primary tumor tissues.
  
  
  RESULTS
  In the pilot testing group, CST6 promoter was methylated in 8/27 (29.6%) operable breast cancer patients, in 6/46 (13.0%) patients with verified metastasis but none of 37 healthy individuals (0%). In the independent cohort, 49/123 (39.8%) operable breast cancer patients were found positive. During the follow up period, 25/123 (20.3%) patients relapsed and 9/123 (7.3%) died. CST6 was methylated in cfDNA of 13/25 (52%) patients that relapsed and in 3/9 (33.3%) patients that died.
  
  
  CONCLUSIONS
  CST6 promoter is highly methylated in cfDNA of breast cancer patients, but not in healthy individuals. CST6 promoter methylation in cfDNA, should be prospectively validated as a novel plasma tumor biomarker for breast cancer in a large cohort of breast cancer patients.
authors.affiliation: Laboratory of Analytical Chemistry, Department of Chemistry, 15771, University of Athens, Greece.
authors.forename: Maria
authors.initials: M
authors.lastname: Chimonidou
authors.forename: Alexandra
authors.initials: A
authors.lastname: Tzitzira
authors.forename: Areti
authors.initials: A
authors.lastname: Strati
authors.forename: Georgia
authors.initials: G
authors.lastname: Sotiropoulou
authors.forename: Costas
authors.initials: C
authors.lastname: Sfikas
authors.forename: Nikos
authors.initials: N
authors.lastname: Malamos
authors.forename: Vasilis
authors.initials: V
authors.lastname: Georgoulias
authors.forename: Evi
authors.initials: E
authors.lastname: Lianidou
mesh_terms: D014408:Biomarkers, Tumor / Q000097:blood*; D001943:Breast Neoplasms / Q000235:genetics* / Q000473:pathology; D016022:Case-Control Studies; D055332:Cystatin M / Q000097:blood / Q000235:genetics*; D019175:DNA Methylation; D004273:DNA, Neoplasm / Q000097:blood*; D005260:Female; D005500:Follow-Up Studies; D006801:Humans; D007150:Immunohistochemistry; D008875:Middle Aged; D009364:Neoplasm Recurrence, Local; D009367:Neoplasm Staging; D009360:Neoplastic Cells, Circulating / Q000473:pathology; D010865:Pilot Projects; D011237:Predictive Value of Tests; D011379:Prognosis; D011401:Promoter Regions, Genetic; D012680:Sensitivity and Specificity
chemical_list: D014408:Biomarkers, Tumor; C525104:CST6 protein, human; D055332:Cystatin M; D004273:DNA, Neoplasm