title: Methylated APC and GSTP1 genes in serum DNA correlate with the presence of circulating blood tumor cells and are associated with a more aggressive and advanced breast cancer disease.
abstract: BACKGROUND
  Tumor-related methylated DNA and circulating tumor cells (CTC) in the peripheral blood might be of prognostic importance in breast cancer. Thus, the aim of our study was to examine free methylated DNA and CTC in the blood from breast cancer patients and to correlate it with clinicopathological features known to influence prognosis.
  
  
  METHODS
  We prospectively obtained serum samples from 85 patients with breast cancer and 22 healthy volunteers. Sera were analysed by methylation specific PCR (MethyLight PCR) for five genes: adenomatous polyposis coli (APC), ras association domain family protein 1A (RASSF1A), estrogen receptor 1 (ESR1), CDKN2A (p16) and glutathione s-transferase pi 1 (GSTP1). Beta actin (ACTB) served as control. In parallel matched peripheral blood of 63 patients was used to assay for circulating tumor cells in the peripheral blood by a modified immunomagnetic AdnaTest BreastCancerSelect with PCR detection for EPCAM, MUC1, MGB1 and SPDEF.
  
  
  RESULTS
  A hypermethylation in the APC gene in 29% (25/85), in RASSF1A in 26% (22/85), in GSTP1 in 18% (14/76) and in ESR1 in 38% (32/85) of all breast cancer patients was detected. No hypermethylation of CDKN2A was found (0/25). Blood samples of patients were defined CTC positive by detecting the EPCAM 13% (8/63), MUC1 16% (10/63), MGB 9% (5/55), SPDEF 12% (7/58) and in 27% detecting one or more genes (15/55). A significant difference was seen in methylated APC DNA between cancer patients and healthy volunteers. Moreover, methylated APC, RASSF1 and CTC were significantly different in metastatic versus non-metastatic disease. In addition, the presence of methylated APC, RASSF1A and CTC correlated significantly with AJCC-staging (p = 0.001, p = 0.031 and 0.002, respectively). High incidences of methylations were found for the genes RASSF1 and ESR1 in healthy individuals (both 23% 5/22). Methylated GSTP1 was predominantly found in the serum of patients with large primaries (p = 0.023) and was highly significantly correlated with positive Her2/neu status (p = 0.003). Elevated serum CA15.3 was strongly correlated with methylated APC and CTC detection (both p = 0.000). Methylated ESR1 failed to exhibit significant correlations with any of the above mentioned parameters. The presence of CTC in peripheral blood was significantly associated with methylated APC (p = 0.012) and methylated GSTP1 (p = 0.001).
  
  
  CONCLUSIONS
  The detection of methylated APC and GSTP1 DNA in serum correlated with the presence of CTC in the blood of breast cancer patients. Both methylated DNA and CTC correlated with a more aggressive tumor biology and advanced disease.
authors.affiliation: Department of Radiation Therapy and Radiation Oncology, University of Düsseldorf, Germany.
authors.forename: C
authors.initials: C
authors.lastname: Matuschek
authors.forename: Edwin
authors.initials: E
authors.lastname: Bölke
authors.forename: G
authors.initials: G
authors.lastname: Lammering
authors.forename: P A
authors.initials: PA
authors.lastname: Gerber
authors.forename: M
authors.initials: M
authors.lastname: Peiper
authors.forename: W
authors.initials: W
authors.lastname: Budach
authors.forename: H
authors.initials: H
authors.lastname: Taskin
authors.forename: H B
authors.initials: HB
authors.lastname: Prisack
authors.forename: G
authors.initials: G
authors.lastname: Schieren
authors.forename: K
authors.initials: K
authors.lastname: Orth
authors.forename: H
authors.initials: H
authors.lastname: Bojar
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D014408:Biomarkers, Tumor / Q000235:genetics*; D001943:Breast Neoplasms / Q000097:blood / Q000175:diagnosis* / Q000235:genetics; D004247:DNA / Q000097:blood; D019175:DNA Methylation; D005260:Female; D017491:Genes, APC; D056426:Genetic Loci; D051549:Glutathione S-Transferase pi / Q000235:genetics*; D006801:Humans; D008875:Middle Aged; D009360:Neoplastic Cells, Circulating; D011379:Prognosis
chemical_list: D014408:Biomarkers, Tumor; D004247:DNA; C496556:GSTP1 protein, human; D051549:Glutathione S-Transferase pi