title: Inconsistent Results With Different Secondary Reflex Assays for Resolving HER2 Status.
abstract: OBJECTIVE
  Guidelines suggest that secondary reflex testing may be useful for resolving HER2 status in breast cancers with equivocal results by both immunohistochemistry (IHC) and in situ hybridization (ISH). We compared two reflex ISH assays and a polymerase chain reaction (PCR) assay for this application.
  
  
  METHODS
  Twenty-nine breast cancers with equivocal IHC and ISH results were retested two ways: (1) ISH using differentially labeled probes targeting ERBB2 ( HER2 , 17q12) and either RAI1 (17p11.2) or ORC4 (2q22.3-2q23.1) in two separate assays and (2) real-time quantitative PCR amplification of ERBB2 and a control locus ( EIF5B , 2q11.2).
  
  
  RESULTS
  Results of the HER2 / RAI1 and HER2 / ORC4 ISH assays were concordant for 21 (72%) cases, and results of all three secondary reflex assays were concordant for only 18 (62%) cases. Result discrepancies between the two ISH readers were observed for cases close to the cutoff threshold.
  
  
  CONCLUSIONS
  Use of different control loci for ISH is associated with discordant results, and PCR is more likely to classify cases as nonamplified, possibly due to contamination with nontumor cells. While resolution of HER2 -equivocal results is desirable from a clinical perspective, different secondary reflex assays yield different results, and the correlation of these results with clinical outcomes is unknown.
authors: From the ARUP Laboratories, Salt Lake City, UT.
authors: Carlynn
authors: C
authors: Willmore-Payne
authors: ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT;
authors: Kristy
authors: K
authors: Damjanovich-Colmenares
authors: ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT;
authors: Alexandra V
authors: AV
authors: Pasi
authors: Huntsman Cancer Institute.
authors: Theresa L
authors: TL
authors: Werner
authors: Department of Pathology, University of Utah, Salt Lake City.
authors: H Evin
authors: HE
authors: Gulbahce
authors: Department of Pathology, University of Utah, Salt Lake City.
authors: Erinn
authors: E
authors: Downs-Kelly
authors: Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN.
authors: Katherine B
authors: KB
authors: Geiersbach
mesh_terms: D014408:Biomarkers, Tumor / Q000378:metabolism*; D001943:Breast Neoplasms / Q000175:diagnosis / Q000378:metabolism* / Q000473:pathology; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D051738:Origin Recognition Complex / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D012018:Reflex; D015534:Trans-Activators; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D014408:Biomarkers, Tumor; D018797:Cell Cycle Proteins; D051738:Origin Recognition Complex; D018719:Erb-b2 Receptor Tyrosine Kinases; D015534:Trans-Activators; D014157:Transcription Factors; C109152:ORC4 protein, human; C431552:RAI1 protein, human; C508053:ERBB2 protein, human
keywords: Breast cancer; HER2; In situ hybridization