title: Comparison of two quantitative polymerase chain reaction methods for detecting HER2/neu amplification.
abstract: Two quantitative polymerase chain reaction (PCR) methods for HER2/neu gene quantification were evaluated for implementation into a clinical laboratory. Assays were developed using sequence-specific hybridization probes to detect a target (HER2/neu) and a reference gene (beta-globin) simultaneously. One method utilizes real-time quantification while the second uses internal competitors and melting curves to quantify the unknown sample. These two methods were evaluated using three cell lines and 97 breast tumor samples. Two hundred ninety-four samples were subsequently evaluated using the real-time quantification and immunohistochemical (IHC) staining. Real-time PCR gave HER2/neu gene doses of 10 for SKBR3 and 2 for T47D while the competitive PCR gave doses of 11 for SKBR3 and 2.2 for T47D. Both methods produced coefficients of variation (CV) of less than 3% for within-run and less than 6% for between-run analysis. Examination of 97 breast tumors found a correlation of r = 0.974 between the two methods. IHC and PCR results agreed for 234 of the subsequent 294 samples analyzed (79% concordance). A subset of ten discrepant samples was microdissected. After microdissection all ten were positive by PCR, thus resolving the discrepancy. Real-time quantification and microdissection is useful clinically for HER2/neu quantification. Its ease of use and broad dynamic range allows screening for amplification of HER2/neu.
authors.affiliation: Associated Regional and University Pathologists Institute for Clinical and Experimental Pathology, Salt Lake City, Utah, USA.
authors.forename: Alison
authors.initials: A
authors.lastname: Millson
authors.forename: Arminda
authors.initials: A
authors.lastname: Suli
authors.forename: Leah
authors.initials: L
authors.lastname: Hartung
authors.forename: Steve
authors.initials: S
authors.lastname: Kunitake
authors.forename: Ann
authors.initials: A
authors.lastname: Bennett
authors.forename: Mary C Lowry
authors.initials: MC
authors.lastname: Nordberg
authors.forename: Wedad
authors.initials: W
authors.lastname: Hanna
authors.forename: Carl T
authors.initials: CT
authors.lastname: Wittwer
authors.forename: Arun
authors.initials: A
authors.lastname: Seth
authors.forename: Elaine
authors.initials: E
authors.lastname: Lyon
mesh_terms: D001483:Base Sequence; D017931:DNA Primers; D018734:Genes, erbB-2; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D017931:DNA Primers