title: Well-differentiated invasive breast cancers with equivocal HER2 immunohistochemistry: what is the yield of routine reflex in-situ hybridization testing?
abstract: OBJECTIVE
  The 2013 American Society of Clinical Oncology and the College of American Pathologists (ASCO/CAP) HER2 guidelines recommend testing all invasive breast cancers for HER2, typically with immunohistochemistry (IHC) followed by in-situ hybridization (ISH) when IHC is equivocal. As well-differentiated breast cancers are rarely HER2-positive, we assessed the value of routine reflex HER2 ISH testing for this subset of breast cancers.
  
  
  RESULTS
  We collected HER2 IHC 2+ cases and fluorescence in-situ hybridization (FISH) data from primary breast cancers with well-differentiated tumour types (grade 1 ductal carcinomas, classic lobular carcinomas, tubular, cribriform and pure mucinous carcinomas) at our centre from 2010 to 2015. Haematoxylin and eosin (H&E) and IHC slides were reviewed to confirm tumour type, grade and IHC score based on ASCO/CAP 2013 guidelines and their recent revisions. Of 4633 invasive carcinomas, 1133 had a well-differentiated tumour type; 177 of these were HER2 IHC equivocal, three of which were low-level amplified by FISH (0.3% of all well-differentiated tumours). One amplified case was classic invasive lobular carcinoma and two were invasive ductal carcinomas, grade 1. One amplified case had chromosome 17 monosomy, and one was rescored as HER2 IHC 1+ upon review. 'Basolateral' staining was noted in one amplified case and in 65 of 174 (37.4%) non-amplified cases. This incomplete membranous staining pattern was observed in the majority of invasive ductal carcinomas that were rescored as 1+ according to the revised 2013 guidelines.
  
  
  CONCLUSIONS
  The rate of HER2 amplification among well-differentiated breast cancers is very low. Basolateral staining in well-differentiated tumours may be overinterpreted as HER2 IHC 2+, but is rarely associated with HER2 amplification.
authors.affiliation: Department of Pathology and Laboratory Medicine, Nova Scotia Health Authority and Dalhousie University, Halifax, Nova Scotia, Canada.
authors.forename: Gillian C
authors.identifier: http://orcid.org/0000-0001-9147-759X
authors.initials: GC
authors.lastname: Bethune
authors.affiliation: Department of Pathology and Laboratory Medicine, Nova Scotia Health Authority and Dalhousie University, Halifax, Nova Scotia, Canada.
authors.forename: Alexandra S L
authors.initials: AS
authors.lastname: Pettit
authors.affiliation: Department of Pathology and Laboratory Medicine, Nova Scotia Health Authority and Dalhousie University, Halifax, Nova Scotia, Canada.
authors.forename: Daniel
authors.initials: D
authors.lastname: Veldhuijzen van Zanten
authors.affiliation: Department of Pathology and Laboratory Medicine, Nova Scotia Health Authority and Dalhousie University, Halifax, Nova Scotia, Canada.
authors.forename: Penelope J
authors.initials: PJ
authors.lastname: Barnes
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D014408:Biomarkers, Tumor / Q000032:analysis*; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D017404:In Situ Hybridization, Fluorescence; D008875:Middle Aged; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000032:analysis*
chemical_list: D014408:Biomarkers, Tumor; D018719:Erb-b2 Receptor Tyrosine Kinases; C508053:ERBB2 protein, human
keywords: ASCO/CAP guidelines; HER2 testing; breast cancer; equivocal category; low grade