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

Abstract

BackgroundHER-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.MethodsTo 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.ResultsThe 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).ConclusionBased 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.

MUC1 oncogene amplification correlates with protein overexpression in invasive breast carcinoma cells

“…Q-PCR has proven to be a very powerful tool to analyze gene copy variations in cancer [25,37]. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 13 By this method, we detected MUC1 gene amplification in 12% of benign lesions and 38% of invasive breast carcinomas.…”

Detection of HER2 Status in Breast Cancer: Comparison of Current Methods with MLPA and Real-time RT-PCR

“…The sensitivity, accuracy and high-throughput potency of qRT-PCR has made it an appropriate candidate for HER2 status determination (Rosa et al, 2009;Baehner et al, 2010). Based on our results, 36% of samples with these samples, one sample presented contrary results (2+ by MLPA, and positive by FISH).…”

“…HER2, as a prognostic and predictive marker, has been widely accepted in the management and treatment of breast cancer (Rosa et al, 2009). The high cost and side effects of trastuzumab therapy demand that highly accurate, robust, sensitive, and cost-effective testing protocols be used in clinical settings (Barberis et al, 2008;Criscitiello and Curigliano, 2013).…”

“…The HER2 (ERBB2) proto-oncogene is located on chromosome 17 and encodes a 185-kD transmembrane tyrosine kinase, which has a critical role in determining patient prognosis and the treatment of breast cancer (O'Malley et al, 2001;Rosa et al, 2009). Approximately or over-expression.…”

Efficacy of Primed In Situ Labelling in Determination of HER-2 Gene Amplification and CEN-17 Status in Breast Cancer Tissue

“…Also the signals produced by the FISH assay decay within a few weeks (Van de Vijver et al, 2007). More recently, the CISH (chromogenic in situ hybridization) methodology, approved by FDA, has emerged as a potential alternative to FISH (Rosa et al 2009). Other new modalities of HER-2 testing are Metallographic In Situ Hybridization (Tubbs et al, 2002; and brightfield double in situ hybridization (Nitta et al, 2008).…”