title: Automated measurement of estrogen receptor in breast cancer: a comparison of fluorescent and chromogenic methods of measurement.
abstract: Whereas FDA-approved methods of assessment of estrogen receptor (ER) are 'fit for purpose', they represent a 30-year-old technology. New quantitative methods, both chromogenic and fluorescent, have been developed and studies have shown that these methods increase the accuracy of assessment of ER. Here, we compare three methods of ER detection and assessment on two retrospective tissue microarray (TMA) cohorts of breast cancer patients: estimates of percent nuclei positive by pathologists and by Aperio's nuclear algorithm (standard chromogenic immunostaining), and immunofluorescence as quantified with the automated quantitative analysis (AQUA) method of quantitative immunofluorescence (QIF). Reproducibility was excellent (R(2)>0.95) between users for both automated analysis methods, and the Aperio and QIF scoring results were also highly correlated, despite the different detection systems. The subjective readings show lower levels of reproducibility and a discontinuous, bimodal distribution of scores not seen by either mechanized method. Kaplan-Meier analysis of 10-year disease-free survival was significant for each method (Pathologist, P=0.0019; Aperio, P=0.0053, AQUA, P=0.0026); however, there were discrepancies in patient classification in 19 out of 233 cases analyzed. Out of these, 11 were visually positive by both chromogenic and fluorescent detection. In 10 cases, the Aperio nuclear algorithm labeled the nuclei as negative; in 1 case, the AQUA score was just under the cutoff for positivity (determined by an Index TMA). In contrast, 8 out of 19 discrepant cases had clear nuclear positivity by fluorescence that was unable to be visualized by chromogenic detection, perhaps because of low positivity masked by the hematoxylin counterstain. These results demonstrate that automated systems enable objective, precise quantification of ER. Furthermore, immunofluorescence detection offers the additional advantage of a signal that cannot be masked by a counterstaining agent. These data support the usage of automated methods for measurement of this and other biomarkers that may be used in companion diagnostic tests.
authors.affiliation: Department of Pathology, Yale University School of Medicine, New Haven, CT, USA.
authors.forename: Elizabeth R
authors.initials: ER
authors.lastname: Zarrella
authors.affiliation: Department of Pathology, Yale University School of Medicine, New Haven, CT, USA.
authors.forename: Madeline
authors.initials: M
authors.lastname: Coulter
authors.affiliation: Department of Pathology, Yale University School of Medicine, New Haven, CT, USA.
authors.forename: Allison W
authors.initials: AW
authors.lastname: Welsh
authors.affiliation: Department of Pathology, Yale University School of Medicine, New Haven, CT, USA.
authors.forename: Daniel E
authors.initials: DE
authors.lastname: Carvajal
authors.affiliation: Department of Pathology, Yale University School of Medicine, New Haven, CT, USA.
authors.forename: Kurt A
authors.initials: KA
authors.lastname: Schalper
authors.affiliation: Department of Pathology, Yale University School of Medicine, New Haven, CT, USA.
authors.forename: Malini
authors.initials: M
authors.lastname: Harigopal
authors.affiliation: Department of Pathology, Yale University School of Medicine, New Haven, CT, USA.
authors.forename: David L
authors.initials: DL
authors.lastname: Rimm
authors.affiliation: Department of Pathology, Yale University School of Medicine, New Haven, CT, USA.
authors.forename: Veronique M
authors.initials: VM
authors.lastname: Neumeister
mesh_terms: D057205:Automation, Laboratory / Q000379:methods; D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology; D002863:Chromogenic Compounds / Q000032:analysis / Q000737:chemistry; D005453:Fluorescence; D005455:Fluorescent Antibody Technique / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis / Q000737:chemistry; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D053208:Kaplan-Meier Estimate; D011379:Prognosis; D011960:Receptors, Estrogen / Q000032:analysis* / Q000737:chemistry; D015203:Reproducibility of Results; D012189:Retrospective Studies; D046888:Tissue Array Analysis / Q000379:methods
chemical_list: D002863:Chromogenic Compounds; D005456:Fluorescent Dyes; D011960:Receptors, Estrogen