title: Concomitant detection of HER2 protein and gene alterations by immunohistochemistry (IHC) and silver enhanced in situ hybridization (SISH) identifies HER2 positive breast cancer with and without gene amplification.
abstract: BACKGROUND
  HER2 status assessment became a mandatory test assay in breast cancer, giving prognostic and predictive information including eligibility for adjuvant anti-HER2 therapy. Precise and reliable assessment of HER2 status is therefore of utmost importance. In this study we analyzed breast cancer samples by a novel technology for concomitant detection of the HER2 protein and gene copy number.
  
  
  METHODS
  Tissue microarrays containing 589 invasive breast cancer samples were analyzed with a double immunohistochemistry (IHC) and silver labeled in situ hybridization (SISH) assay simultaneously detecting HER2 protein and gene copy number in the same tumor cells. This bright-field assay was analyzed using scores according to the modified ASCO guidelines and the results were correlated with patient prognosis.
  
  
  RESULTS
  Overall concordance rate between protein expression and the presence of gene amplification was 98%. Fifty-seven of 60 tumors (95%) with IHC score 3+, 6 of 10 tumors with IHC score 2+ (60%) and only 3 of 519 tumors (0.6%) with IHC score 0/1+ were amplified by SISH. Patients with gene amplification despite IHC score 0/1+ had a tendency for worse overall survival (p = 0.088, reaching nearly statistical significance) compared to IHC score 0/1+ without amplification. In contrast, there was no difference in overall survival in IHC score 3+/2+ tumors with and without gene amplification.
  
  
  CONCLUSIONS
  The novel double IHC and SISH assay for HER2 is efficient in the identification of breast cancer with discordant HER2 protein and HER2 gene status, especially for the prognostically relevant groups of HER2 protein negative tumors with HER2 amplification and HER2 protein positive tumors without HER2 amplification. Breast cancer without HER2 amplification among IHC score 2+/3+ tumors (10% in our cohort) suggests that other mechanisms than gene amplification contribute to protein overexpression in these cells.
authors.affiliation: Institute of Surgical Pathology, University Hospital Zurich, Zurich, Switzerland.
authors.forename: Zsuzsanna
authors.initials: Z
authors.lastname: Varga
authors.affiliation: Section of Molecular Pathology, Cleveland Clinic, Cleveland, Ohio, United States of America.
authors.forename: Raymond R
authors.initials: RR
authors.lastname: Tubbs
authors.affiliation: Institute of Surgical Pathology, University Hospital Zurich, Zurich, Switzerland.
authors.forename: Holger
authors.initials: H
authors.lastname: Moch
mesh_terms: D001943:Breast Neoplasms / Q000175:diagnosis* / Q000235:genetics / Q000378:metabolism; D005260:Female; D005784:Gene Amplification; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D017403:In Situ Hybridization / Q000379:methods*; D011379:Prognosis; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D046888:Tissue Array Analysis
chemical_list: D018719:Erb-b2 Receptor Tyrosine Kinases; C508053:ERBB2 protein, human