title: Evaluation of HER2 Gene Status in Breast Cancer Samples with Indeterminate Fluorescence in Situ Hybridization by Quantitative Real-Time PCR.
abstract: Administration of drugs targeting HER2 (official symbol ERBB2) is an important component of therapy for breast cancer patients with HER2 amplification/overexpression as determined by in situ hybridization (ISH) and immunohistochemistry (IHC). In approximately 5% of breast cancers, ISH assays fail. In these cases, HER2 protein expression is evaluated by IHC alone that may yield false negatives/positives for poor-quality samples. Therefore, we developed a method that was based on quantitative real-time PCR applicable for DNA from formalin-fixed, paraffin-embedded tissue samples. Its limit of detection was determined with breast cancer cell lines and validated with 223 breast cancer patient samples. On the basis of comparisons with fluorescent ISH (FISH) and IHC data, the sensitivity of the new method was 94.2% and 95.1%, its specificity was 100% and 99.1%, and overall concordance between results obtained with the quantitative real-time PCR method and FISH/IHC was 97.6% for both methods. The quantitative real-time PCR method was then used to evaluate the HER2 status of 198 of 3696 breast cancer tissues that yielded indeterminate FISH results. The HER2 copy number was successfully determined in 69.2% of these indeterminate samples. Thus, the DNA-based technique appears to be a specific, sensitive method for determining HER2 copy numbers when the FISH assay fails, which may complement IHC tests.
authors.affiliation: Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University and University Hospital in Olomouc, Olomouc, Czech Republic.
authors.forename: Vladimira
authors.initials: V
authors.lastname: Koudelakova
authors.affiliation: Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University and University Hospital in Olomouc, Olomouc, Czech Republic.
authors.forename: Jitka
authors.initials: J
authors.lastname: Berkovcova
authors.affiliation: Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University and University Hospital in Olomouc, Olomouc, Czech Republic.
authors.forename: Radek
authors.initials: R
authors.lastname: Trojanec
authors.affiliation: Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University and University Hospital in Olomouc, Olomouc, Czech Republic.
authors.forename: Jana
authors.initials: J
authors.lastname: Vrbkova
authors.affiliation: Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University and University Hospital in Olomouc, Olomouc, Czech Republic.
authors.forename: Lenka
authors.initials: L
authors.lastname: Radova
authors.affiliation: Department of Clinical and Molecular Pathology, Faculty of Medicine and Dentistry, Palacky University and University Hospital in Olomouc, Olomouc, Czech Republic.
authors.forename: Jiri
authors.initials: J
authors.lastname: Ehrmann
authors.affiliation: Department of Clinical and Molecular Pathology, Faculty of Medicine and Dentistry, Palacky University and University Hospital in Olomouc, Olomouc, Czech Republic.
authors.forename: Zdenek
authors.initials: Z
authors.lastname: Kolar
authors.affiliation: Department of Oncology, Faculty of Medicine and Dentistry, Palacky University and University Hospital in Olomouc, Olomouc, Czech Republic.
authors.forename: Bohuslav
authors.initials: B
authors.lastname: Melichar
authors.affiliation: Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University and University Hospital in Olomouc, Olomouc, Czech Republic. Electronic address: marian.hajduch@upol.cz.
authors.forename: Marian
authors.initials: M
authors.lastname: Hajduch
mesh_terms: D014408:Biomarkers, Tumor / Q000032:analysis*; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D005260:Female; D005557:Formaldehyde / Q000737:chemistry; D005784:Gene Amplification; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007124:Immunoenzyme Techniques; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D016612:Paraffin Embedding / Q000379:methods; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism*; D012189:Retrospective Studies; D014407:Tumor Cells, Cultured
chemical_list: D014408:Biomarkers, Tumor; D005557:Formaldehyde; D018719:Erb-b2 Receptor Tyrosine Kinases; C508053:ERBB2 protein, human