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

• Published in: BMC cancer Vol. 9; no. 1; p. 90
• Main Authors: Rosa, Fabíola E, Silveira, Sara M, Silveira, Cássia G T, Bérgamo, Nádia A, Neto, Francisco A Moraes, Domingues, Maria A C, Soares, Fernando A, Caldeira, José R F, Rogatto, Silvia R
• Format: Journal Article
• Language: English
• Published: England BioMed Central Ltd 23.03.2009; BioMed Central; BMC
• Subjects: Adult; Aged; Aged, 80 and over; Breast cancer; Breast Neoplasms - genetics; Breast Neoplasms - metabolism; Breast Neoplasms - pathology; Diagnosis; Drug therapy; Female; Gene Amplification; Gene Dosage; Genetic aspects; Health aspects; Humans; Immunohistochemistry; In situ hybridization; In Situ Hybridization, Fluorescence - methods; Middle Aged; Neoplasm Staging; Polymerase chain reaction; Receptor, ErbB-2 - genetics; Receptor, ErbB-2 - metabolism; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction - methods; Brazil
• ISSN: 1471-2407; 1471-2407
• DOI: 10.1186/1471-2407-9-90

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.