title: Standardization of estrogen receptor measurement in breast cancer suggests false-negative results are a function of threshold intensity rather than percentage of positive cells.
abstract: OBJECTIVE
  Recent misclassification (false negative) incidents have raised awareness concerning limitations of immunohistochemistry (IHC) in assessment of estrogen receptor (ER) in breast cancer. Here we define a new method for standardization of ER measurement and then examine both change in percentage and threshold of intensity (immunoreactivity) to assess sources for test discordance.
  
  
  METHODS
  An assay was developed to quantify ER by using a control tissue microarray (TMA) and a series of cell lines in which ER immunoreactivity was analyzed by quantitative immunoblotting in parallel with the automated quantitative analysis (AQUA) method of quantitative immunofluorescence (QIF). The assay was used to assess the ER protein expression threshold in two independent retrospective cohorts from Yale and was compared with traditional methods.
  
  
  RESULTS
  Two methods of analysis showed that change in percentage of positive cells from 10% to 1% did not significantly affect the overall number of ER-positive patients. The standardized assay for ER on two Yale TMA cohorts showed that 67.9% and 82.5% of the patients were above the 2-pg/μg immunoreactivity threshold. We found 9.1% and 19.7% of the patients to be QIF-positive/IHC-negative, and 4.0% and 0.4% to be QIF-negative/IHC-positive for a total of 13.1% and 20.1% discrepant cases when compared with pathologists' judgment of threshold. Assessment of survival for both cohorts showed that patients who were QIF-positive/pathologist-negative had outcomes similar to those of patients who had positive results for both assays.
  
  
  CONCLUSIONS
  Assessment of intensity threshold by using a quantitative, standardized assay on two independent cohorts suggests discordance in the 10% to 20% range with current IHC methods, in which patients with discrepant results have prognostic outcomes similar to ER-positive patients with concordant results.
authors.affiliation: Department of Pathology, BML 116, Yale University School of Medicine, 310 Cedar St, PO Box 208023, New Haven, CT, USA.
authors.forename: Allison W
authors.initials: AW
authors.lastname: Welsh
authors.forename: Christopher B
authors.initials: CB
authors.lastname: Moeder
authors.forename: Sudha
authors.initials: S
authors.lastname: Kumar
authors.forename: Peter
authors.initials: P
authors.lastname: Gershkovich
authors.forename: Elaine T
authors.initials: ET
authors.lastname: Alarid
authors.forename: Malini
authors.initials: M
authors.lastname: Harigopal
authors.forename: Bruce G
authors.initials: BG
authors.lastname: Haffty
authors.forename: David L
authors.initials: DL
authors.lastname: Rimm
mesh_terms: D014408:Biomarkers, Tumor / Q000378:metabolism*; D001943:Breast Neoplasms / Q000378:metabolism*; D018270:Carcinoma, Ductal, Breast / Q000378:metabolism*; D045744:Cell Line, Tumor; D005188:False Negative Reactions; D005260:Female; D005455:Fluorescent Antibody Technique; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D011960:Receptors, Estrogen / Q000378:metabolism*
chemical_list: D014408:Biomarkers, Tumor; D011960:Receptors, Estrogen