title: A single-tube quantitative assay for mRNA levels of hormonal and growth factor receptors in breast cancer specimens.
abstract: Knowledge of estrogen receptor (ER) and progesterone receptor (PR) status has been critical in the evolution of modern targeted therapy of breast cancer and remains essential for making informed therapeutic decisions. Recently, growth factor receptor HER2/neu (ERBB2) status has made it possible to provide another form of targeted therapy linked to the overexpression of this protein. Presently, pathologists determine the receptor status in formalin-fixed, paraffin-embedded sections using subjective, semiquantitative immunohistochemistry (IHC) assays and quantitative fluorescence in situ hybridization for HER2. We developed a single-tube multiplex TaqMan (mERPR+HER2) assay to quantitate mRNA levels of ER, PR, HER2, and two housekeeping genes for breast cancer formalin-fixed, paraffin-embedded sections. Using data from the discovery sample sets, we evaluated IHC-status-dependent cutoff-point and IHC-status-independent clustering methods for the classification of receptor status and then validated these results with independent sample sets. Compared with IHC-status, the accuracies of the mERPR+HER2 assay with the cutoff-point classification method were 0.98 (95% CI: 0.97-1.00), 0.92 (95% CI: 0.88-0.95), and 0.97 (95% CI: 0.95-0.99) for ER, PR, and HER2, respectively, for the validation sets. Furthermore, the areas under the receiver operating-characteristic curves were 0.997 (95% CI: 0.994-1.000), 0.967 (95% CI: 0.949-0.985), and 0.968 (95% CI: 0.915-1.000) for ER, PR, and HER2, respectively. This multiplex assay provides a sensitive and reliable method to quantitate hormonal and growth factor receptors.
authors.affiliation: Breast Pathology Laboratory, Guy's Hospital, London, United Kingdom.
authors.forename: Ayuko A
authors.initials: AA
authors.lastname: Iverson
authors.forename: Cheryl
authors.initials: C
authors.lastname: Gillett
authors.forename: Paul
authors.initials: P
authors.lastname: Cane
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Santini
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Vess
authors.forename: Lauren
authors.initials: L
authors.lastname: Kam-Morgan
authors.forename: Alice
authors.initials: A
authors.lastname: Wang
authors.forename: Marcia
authors.initials: M
authors.lastname: Eisenberg
authors.forename: Charles M
authors.initials: CM
authors.lastname: Rowland
authors.forename: Janice J
authors.initials: JJ
authors.lastname: Hessling
authors.forename: Samuel E
authors.initials: SE
authors.lastname: Broder
authors.forename: John J
authors.initials: JJ
authors.lastname: Sninsky
authors.forename: Andrew
authors.initials: A
authors.lastname: Tutt
authors.forename: Steven
authors.initials: S
authors.lastname: Anderson
authors.forename: Sheng-Yung P
authors.initials: SY
authors.lastname: Chang
mesh_terms: D001943:Breast Neoplasms / Q000175:diagnosis*; D005260:Female; D005557:Formaldehyde / Q000737:chemistry; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D016612:Paraffin Embedding; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics*; D011960:Receptors, Estrogen / Q000235:genetics*; D011980:Receptors, Progesterone / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D016707:Tissue Fixation; D015854:Up-Regulation
chemical_list: D005557:Formaldehyde; D012333:RNA, Messenger; D018719:Erb-b2 Receptor Tyrosine Kinases; D011960:Receptors, Estrogen; D011980:Receptors, Progesterone