title: Noninvasive detection of HER2 amplification with plasma DNA digital PCR.
abstract: OBJECTIVE
  Digital PCR is a highly accurate method of determining DNA concentration. We adapted digital PCR to determine the presence of oncogenic amplification through noninvasive analysis of circulating free plasma DNA and exemplify this approach by developing a plasma DNA digital PCR assay for HER2 copy number.
  
  
  METHODS
  The reference gene for copy number assessment was assessed experimentally and bioinformatically. Chromosome 17 pericentromeric probes were shown to be suboptimal, and EFTUD2 at chromosome position 17q21.31 was selected for analysis. Digital PCR assay parameters were determined on plasma samples from a development cohort of 65 patients and assessed in an independent validation cohort of plasma samples from 58 patients with metastatic breast cancer. The sequential probability ratio test was used to assign the plasma DNA digital PCR test as being HER2-positive or -negative in the validation cohort.
  
  
  RESULTS
  In the development cohort, the HER2:EFTUD2 plasma DNA copy number ratio had a receiver operator area under the curve (AUC) = 0.92 [95% confidence interval (CI), 0.86-0.99, P = 0.0003]. In the independent validation cohort, 64% (7 of 11) of patients with HER2-amplified cancers were classified as plasma digital PCR HER2-positive and 94% (44 of 47) of patients with HER2-nonamplified cancers were classified as digital PCR HER2-negative, with a positive and negative predictive value of 70% and 92%, respectively.
  
  
  CONCLUSIONS
  Analysis of plasma DNA with digital PCR has the potential to screen for the acquisition of HER2 amplification in metastatic breast cancer. This approach could potentially be adapted to the analysis of any locus amplified in cancer.
authors: The Breakthrough Breast Cancer Research Centre, Institute of Cancer Research, London, United Kingdom.
authors: Heidrun
authors: H
authors: Gevensleben
authors: Isaac
authors: I
authors: Garcia-Murillas
authors: Monika K
authors: MK
authors: Graeser
authors: Gaia
authors: G
authors: Schiavon
authors: Peter
authors: P
authors: Osin
authors: Marina
authors: M
authors: Parton
authors: Ian E
authors: IE
authors: Smith
authors: Alan
authors: A
authors: Ashworth
authors: Nicholas C
authors: NC
authors: Turner
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001943:Breast Neoplasms / Q000097:blood / Q000235:genetics* / Q000473:pathology; D002886:Chromosomes, Human, Pair 17; D056915:DNA Copy Number Variations / Q000235:genetics; D004273:DNA, Neoplasm / Q000097:blood; D005260:Female; D006801:Humans; D008875:Middle Aged; D009362:Neoplasm Metastasis / Q000235:genetics / Q000473:pathology; D010445:Peptide Elongation Factors / Q000097:blood* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000097:blood* / Q000302:isolation & purification; D017415:Ribonucleoprotein, U5 Small Nuclear
chemical_list: D004273:DNA, Neoplasm; D010445:Peptide Elongation Factors; D018719:Erb-b2 Receptor Tyrosine Kinases; D017415:Ribonucleoprotein, U5 Small Nuclear; C581608:EFTUD2 protein, human; C508053:ERBB2 protein, human
references: Proc Natl Acad Sci U S A. 1999 Aug 3;96(16):9236-41
references: 10430926
references: Breast Cancer Res Treat. 2012 Jul;134(1):283-9
references: 22476856
references: Sci Transl Med. 2012 Nov 28;4(162):162ra154
references: 23197571
references: N Engl J Med. 2001 Mar 15;344(11):783-92
references: 11248153
references: PLoS One. 2011 Jan 10;6(1):e15624
references: 21264346
references: Anal Chem. 2011 Nov 15;83(22):8604-10
references: 22035192
references: Proc Natl Acad Sci U S A. 2007 Aug 7;104(32):13116-21
references: 17664418
references: Lancet. 2010 Aug 28;376(9742):687-97
references: 20728210
references: Nature. 2010 Oct 28;467(7319):1109-13
references: 20981101
references: Nat Methods. 2006 Feb;3(2):95-7
references: 16432518
references: Clin Cancer Res. 2005 Sep 15;11(18):6598-607
references: 16166438
references: Nat Biotechnol. 2001 Jan;19(1):78-81
references: 11135558
references: Breast Cancer Res Treat. 2011 Oct;129(3):659-74
references: 21698410
references: Breast Cancer Res Treat. 2010 Apr;120(2):461-7
references: 20107891
references: Nature. 2012 Oct 4;490(7418):61-70
references: 23000897
references: Nat Methods. 2006 Jul;3(7):551-9
references: 16791214
references: Nature. 2010 Apr 15;464(7291):999-1005
references: 20393555
references: Br J Cancer. 2011 Apr 12;104(8):1342-8
references: 21427727
references: Proc Natl Acad Sci U S A. 2011 Sep 13;108(37):E699-708
references: 21876152
references: Breast Cancer Res. 2012 Mar 20;14(2):R53
references: 22433433
references: Lancet Oncol. 2012 Apr;13(4):e178-85
references: 22469128
references: Clin Chem. 2002 Aug;48(8):1186-93
references: 12142371
references: Clin Cancer Res. 2009 Jan 15;15(2):441-51
references: 19147748
references: J Clin Oncol. 2007 Jan 1;25(1):118-45
references: 17159189
references: Breast Cancer Res Treat. 2010 Nov;124(2):403-12
references: 20859679
references: Breast Cancer Res. 2010;12(6):R92
references: 21059212
references: Cancer Cell. 2010 Jan 19;17(1):77-88
references: 20129249
references: Nat Med. 2008 Sep;14(9):985-90
references: 18670422
references: Genome Res. 2012 Feb;22(2):220-31
references: 21990379