title: Circulating cell-free DNA: a promising marker of regional lymphonode metastasis in breast cancer patients.
abstract: OBJECTIVE
  We undertook the current study with untreated breast cancer to (1) role the variations in the plasma levels of cfDNA and the size distribution in early stage, (2) determine the frequency in plasma of methylation of three candidate genes, RASSF1A, MAL, and SFRP1, and (3) to determine whether detection of cfDNA variations and methylation changes in plasma might have specific clinical utility.
  
  
  METHODS
  Thirty-nine patients woman patients (median age 64 years; range, 36-90 years) who underwent surgery for primary BR and 49 healthy females' subjects (control group without any breast lesion) were evaluated. The cfDNA levels were analyzed using quantitative real-time polymerase chain reaction of β-globin. Based on the ALU repeats, the cfDNA was considered as either total (fragments of 115 bp, ALU115) or tumoral (fragments of 247 bp, ALU247). The association between the levels of the ALU247, ALU115 repeat, and ALU 247/115and the pathologic tumor characteristics was analyzed. Used methylight qPCR method, cfDNA from plasma samples of healthy donors and patients with breast cancer were evaluated for the diagnotic value of the methylation status of three genes (RASSF1A, MAL, SFRP1) frequently methylated in breast cancer.
  
  
  RESULTS
  The baseline levels of cfDNA were significantly higher in the patients with cancer, and the level of ALU247 was the most accurate circulating cfDNA marker in discriminating the cancer from non-cancer subjects. A high statistical significance was found by considering the T stage and patients with regional LN metastasis positive cancers showed significantly higher cfDNA level of ALU247. Moreover, patients with methylation of at least one of the gene under investigate showed a higher quantity of cfDNA ALU115 (p< 0.0001) and ALU247 level (p< 0.0001).
  
  
  CONCLUSIONS
  We observed that necrosis could be a potential source of circulating tumour-specific cfDNA ALU247; and that cfDNA ALU247 and methylated cfDNA (RASSF1A, MAL and SFRP1) are both a phenotypic feature of tumour biology.
authors.affiliation: Department of Surgical, Oncological and Gastroenterological Sciences, 2nd Surgical Clinic, Padova, Italy. m.agostini@unipd.it
authors.forename: M
authors.initials: M
authors.lastname: Agostini
authors.forename: M V
authors.initials: MV
authors.lastname: Enzo
authors.forename: C
authors.initials: C
authors.lastname: Bedin
authors.forename: V
authors.initials: V
authors.lastname: Belardinelli
authors.forename: E
authors.initials: E
authors.lastname: Goldin
authors.forename: P
authors.initials: P
authors.lastname: Del Bianco
authors.forename: E
authors.initials: E
authors.lastname: Maschietto
authors.forename: E
authors.initials: E
authors.lastname: D'Angelo
authors.forename: Leo
authors.initials: L
authors.lastname: Izzi
authors.forename: A
authors.initials: A
authors.lastname: Saccani
authors.forename: G
authors.initials: G
authors.lastname: Zavagno
authors.forename: D
authors.initials: D
authors.lastname: Nitti
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D014408:Biomarkers, Tumor / Q000097:blood / Q000235:genetics*; D001943:Breast Neoplasms / Q000097:blood / Q000235:genetics* / Q000473:pathology*; D016022:Case-Control Studies; D004270:DNA, Circular / Q000097:blood / Q000235:genetics*; D004273:DNA, Neoplasm / Q000097:blood* / Q000235:genetics; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics; D008198:Lymph Nodes / Q000473:pathology*; D008207:Lymphatic Metastasis; D008565:Membrane Proteins / Q000235:genetics; D008875:Middle Aged; D062806:Myelin and Lymphocyte-Associated Proteolipid Proteins / Q000235:genetics; D025521:Tumor Suppressor Proteins / Q000235:genetics; D000094742:Secreted Frizzled-Related Proteins
chemical_list: D014408:Biomarkers, Tumor; D004270:DNA, Circular; D004273:DNA, Neoplasm; D036341:Intercellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D062806:Myelin and Lymphocyte-Associated Proteolipid Proteins; D025521:Tumor Suppressor Proteins; C569026:MAL protein, human; C411762:RASSF1 protein, human; C117636:SFRP1 protein, human; D000094742:Secreted Frizzled-Related Proteins