title: Real-time quantitative PCR of microdissected paraffin-embedded breast carcinoma: an alternative method for HER-2/neu analysis.
abstract: We studied the feasibility of using real-time quantitative PCR to determine HER-2 DNA amplification and mRNA expression in microdissected formalin-fixed, paraffin-embedded breast tumors and compared this with standard immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH) methods. Study cases (27 carcinomas and 3 ductal breast carcinoma in situ (DCIS) cases) showed varying Her-2 expression as determined by IHC (HercepTest). In carcinomas, there was a good correlation between HER-2 DNA amplification and strong HER-2 protein expression detected by FISH and IHC, respectively. A single DCIS case was amplified in FISH, but not in IHC. Both HER-2 gene amplification and expression could be quantified in microdissected paraffin-embedded tumors using real-time PCR, DNA and RNA being successfully detected in 146 of 150 (97%) and 141 of 150 (94%) samples, respectively. PCR analysis for HER-2 DNA amplification using the LightCycler HER2/neu DNA Quantification kit (Roche Molecular Biochemicals, Mannheim, Germany) correlated fairly well with IHC and FISH. All IHC HER-2 3+ tumors were amplified according to the kit, as was the FISH-amplified DCIS case. DNA-PCR identified five additional tumors as being amplified. Interestingly, all these scored 2+ with the HercepTest, but were negative using FISH. We believe that real-time quantitative PCR analysis of HER-2 DNA amplification following microdissection represents a useful supplementary or perhaps even an alternative technique for establishing HER-2 status in paraffin-embedded tumors.
authors.affiliation: Institutes of Pathology, Aarhus University Hospital, Aarhus, Denmark. lmgje@mail.dk
authors.forename: Lise Mette
authors.initials: LM
authors.lastname: Gjerdrum
authors.forename: Boe Sandahl
authors.initials: BS
authors.lastname: Sorensen
authors.forename: Eigil
authors.initials: E
authors.lastname: Kjeldsen
authors.forename: Flemming Brandt
authors.initials: FB
authors.lastname: Sorensen
authors.forename: Ebba
authors.initials: E
authors.lastname: Nexo
authors.forename: Stephen
authors.initials: S
authors.lastname: Hamilton-Dutoit
mesh_terms: D001943:Breast Neoplasms / Q000235:genetics* / Q000473:pathology; D004273:DNA, Neoplasm / Q000235:genetics / Q000378:metabolism; D005260:Female; D005784:Gene Amplification; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D017404:In Situ Hybridization, Fluorescence / Q000379:methods; D008867:Microtomy / Q000379:methods; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000032:analysis / Q000235:genetics*; D015203:Reproducibility of Results; D016610:Tissue Embedding
chemical_list: D004273:DNA, Neoplasm; D012333:RNA, Messenger; D018719:Erb-b2 Receptor Tyrosine Kinases