title: Quantitative real-time RT-PCR and chromogenic in situ hybridization: precise methods to detect HER-2 status in breast carcinoma.
abstract: BACKGROUND
  HER-2 gene testing has become an integral part of breast cancer patient diagnosis. The most commonly used assay in the clinical setting for evaluating HER-2 status is immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). These procedures permit correlation between HER-2 expression and morphological features. However, FISH signals are labile and fade over time, making post-revision of the tumor difficult. CISH (chromogenic in situ hybridization) is an alternative procedure, with certain advantages, although still limited as a diagnostic tool in breast carcinomas.
  
  
  METHODS
  To elucidate the molecular profile of HER-2 status, mRNA and protein expression in 75 invasive breast carcinomas were analyzed by real time quantitative RT-PCR (qRT-PCR) and IHC, respectively. Amplifications were evaluated in 43 of these cases by CISH and in 11 by FISH.
  
  
  RESULTS
  The concordance rate between IHC and qRT-PCR results was 78.9%, and 94.6% for qRT-PCR and CISH. Intratumoral heterogeneity of HER-2 status was identified in three cases by CISH. The results of the three procedures were compared and showed a concordance rate of 83.8%; higher discordances were observed in 0 or 1+ immunostaining cases, which showed high-level amplification (15.4%) and HER-2 transcript overexpression (20%). Moreover, 2+ immunostaining cases presented nonamplified status (50%) by CISH and HER-2 downexpression (38.5%) by qRT-PCR. In general, concordance occurred between qRT-PCR and CISH results. A high concordance was observed between CISH/qRT-PCR and FISH. Comparisons with clinicopathological data revealed a significant association between HER-2 downexpression and the involvement of less than four lymph nodes (P = 0.0350).
  
  
  CONCLUSIONS
  Based on these findings, qRT-PCR was more precise and reproducible than IHC. Furthermore, CISH was revealed as an alternative and useful procedure for investigating amplifications involving the HER-2 gene.
authors.affiliation: Department of Genetics, Institute of Biosciences, UNESP, São Paulo State University, Botucatu, Sao Paulo, Brazil. fabiolarosa7@yahoo.com.br
authors.forename: Fabíola E
authors.initials: FE
authors.lastname: Rosa
authors.forename: Sara M
authors.initials: SM
authors.lastname: Silveira
authors.forename: Cássia G T
authors.initials: CG
authors.lastname: Silveira
authors.forename: Nádia A
authors.initials: NA
authors.lastname: Bérgamo
authors.forename: Francisco A Moraes
authors.initials: FA
authors.lastname: Neto
authors.forename: Maria A C
authors.initials: MA
authors.lastname: Domingues
authors.forename: Fernando A
authors.initials: FA
authors.lastname: Soares
authors.forename: José R F
authors.initials: JR
authors.lastname: Caldeira
authors.forename: Silvia R
authors.initials: SR
authors.lastname: Rogatto
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D005260:Female; D005784:Gene Amplification; D018628:Gene Dosage; D006801:Humans; D007150:Immunohistochemistry; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008875:Middle Aged; D009367:Neoplasm Staging; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
chemical_list: D018719:Erb-b2 Receptor Tyrosine Kinases