title: Detection of HER2 amplification in circulating free DNA in patients with breast cancer.
abstract: BACKGROUND
  Human epidermal growth factor receptor 2 (HER2) is amplified and overexpressed in 20-25% of breast cancers. This study investigated circulating free DNA (cfDNA) for detection of HER2 gene amplification in patients with breast cancer.
  
  
  METHODS
  Circulating free DNA was extracted from plasma of unselected patients with primary breast cancer (22 before surgery and 68 following treatment), 30 metastatic patients and 98 female controls using the QIAamp Blood DNA Mini Kit (Qiagen). The ratio of HER2 to an unamplified reference gene (contactin-associated protein 1 (CNTNAP1)) was measured in cfDNA samples by quantitative PCR (qPCR) using SK-BR-3 cell line DNA as a positive control.
  
  
  RESULTS
  We validated the qPCR assay with DNA extracted from 23 HER2 3+ and 40 HER2-negative tumour tissue samples; the results agreed for 60 of 63 (95.2%) tumours. Amplification was detected in cfDNA for 8 of 68 patients following primary breast cancer treatment and 5 of 30 metastatic patients, but was undetected in 22 patients with primary breast cancer and 98 healthy female controls. Of the patients with amplification in cfDNA, 10 had HER2 3+ tumour status by immunohistochemistry.
  
  
  CONCLUSIONS
  The results demonstrate for the first time the existence of amplified HER2 in cfDNA in the follow-up of breast cancer patients who are otherwise disease free. This approach could potentially provide a marker in patients with HER2-positive breast cancer.
authors.affiliation: Department of Cancer Studies and Molecular Medicine, University of Leicester, Robert Kilpatrick Building, PO Box 65, Leicester Royal Infirmary, Leicester LE2 7LX, UK.
authors.forename: K
authors.initials: K
authors.lastname: Page
authors.forename: N
authors.initials: N
authors.lastname: Hava
authors.forename: B
authors.initials: B
authors.lastname: Ward
authors.forename: J
authors.initials: J
authors.lastname: Brown
authors.forename: D S
authors.initials: DS
authors.lastname: Guttery
authors.forename: C
authors.initials: C
authors.lastname: Ruangpratheep
authors.forename: K
authors.initials: K
authors.lastname: Blighe
authors.forename: A
authors.initials: A
authors.lastname: Sharma
authors.forename: R A
authors.initials: RA
authors.lastname: Walker
authors.forename: R C
authors.initials: RC
authors.lastname: Coombes
authors.forename: J A
authors.initials: JA
authors.lastname: Shaw
mesh_terms: D001943:Breast Neoplasms / Q000097:blood / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D002277:Carcinoma / Q000097:blood / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D016022:Case-Control Studies; D045744:Cell Line, Tumor; D004247:DNA / Q000032:analysis / Q000097:blood*; D004252:DNA Mutational Analysis; D005260:Female; D005500:Follow-Up Studies; D005784:Gene Amplification; D006801:Humans; D009362:Neoplasm Metastasis; D010641:Phenotype; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000097:blood / Q000235:genetics* / Q000378:metabolism; D012189:Retrospective Studies
chemical_list: D004247:DNA; D018719:Erb-b2 Receptor Tyrosine Kinases; C508053:ERBB2 protein, human