Open Access Highly Accessed Research article

Quantitative real-time RT-PCR and chromogenic in situ hybridization: precise methods to detect HER-2 status in breast carcinoma

Fabíola E Rosa1, Sara M Silveira1, Cássia GT Silveira1, Nádia A Bérgamo27, Francisco A Moraes Neto3, Maria AC Domingues4, Fernando A Soares5, José RF Caldeira6 and Silvia R Rogatto27*

Author Affiliations

1 Department of Genetics, Institute of Biosciences, UNESP – São Paulo State University, Botucatu, Sao Paulo, Brazil

2 Department of Urology, Faculty of Medicine, UNESP – São Paulo State University, Botucatu, Brazil

3 Department of Pathology, Amaral Carvalho Hospital, Jaú, São Paulo, Brazil

4 Department of Pathology, Faculty of Medicine, UNESP – São Paulo State University, Botucatu, São Paulo, Brazil

5 Department of Pathology, Fundação Antonio Prudente, Hospital AC Camargo, São Paulo, Brazil

6 Department of Senology, Amaral Carvalho Hospital, Jaú, São Paulo, Brazil

7 NeoGene Laboratory, Fundação Antonio Prudente 211, Hospital AC Camargo – Liberdade São Paulo, Brazil

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BMC Cancer 2009, 9:90  doi:10.1186/1471-2407-9-90

Published: 23 March 2009



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.


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.


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).


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.