Comparative evaluation of non-informative HER-2 immunoreactions (2+) in breast carcinomas with FISH, CISH and QRT-PCR

Abstract

The routine assessment of HER-2 expression can be affected by many immunohistological preanalytical and analytical variables. The evaluation of non-informative HER-2 tests, because of 2(+) scores, has been addressed in studies using in situ hybridization (fluorescent in situ hybridization (FISH) or chromogenic in situ hybridization (CISH)). There are very few studies that additionally checked 2(+) cases by quantitative reverse transcription-PCR (QRT-PCR). We analyzed totally 195 breast carcinoma cases, 70 of them showing 2(+) immunoreaction, with FISH/CISH and QRT-PCR. Confirmed amplification in 2(+) cases fell within the reported range (12.8% vs. 8-44%) and some of them showed lower mRNA levels indicating a genuine decrease of HER-2 protein as a mechanism for the non-informative score. In other cases, increased mRNA levels could be ascribed to HER-2 polysomy, verifying previous observations of immunohistologically detectable HER-2 polysomy. A remarkable subset of the 2(+) cases showed "normal" mRNA levels without amplification or polysomy and technical parameters as well as heterogeneity could be incriminated. The overall concordance of QRT-PCR and FISH was 93.8%, highest than most previously reported. Yet, the lack of clear cut-off mRNA values and the challenge of sample microdissection hinder QRT-PCR from claiming the status of a gold standard test for HER-2 evaluation.

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

“…We observed high agreement between qRT-PCR and FISH, similar to some other studies (Kostopoulou et al, 2007;Baehner et al, 2010). The qRT-PCR method was positive by both FISH and MLPA, while one sample with overexpression detected by qRT-PCR had negative results in the other two tests (Table 3).…”

Comparison of fluorescence in situ hybridization and immunohistochemistry for assessment of HER-2 status in breast cancer patients

“…Kostopoulou et al [7] also confirmed that mRNA levels of these cases with "non-informative" 2+ score were lower than those recorded as high-level amplification, and further supported the notion that in these cases there was a relative reduction of protein expression despite HER-2 amplification. They also found that in almost half of those 2+ score cases, increased mRNA levels could be ascribed to HER-2 polysomy, verifying previous observations of immunohistologically detectable HER-2 polysomy [11] .…”

“…This problem has not been addressed specifically in many studies although it is common in practice. Kostopoulou et al [7] reviewed 13 studies in which the cases with IHC score 2+ were confirmed with FISH, and showed that the average percentage of the cases both with IHC score 2+ and HER-2 amplification was 21.2%, with a range of 8%-44.4%. A study in Taiwan had demonstrated that 53% of the IHC 2+ cases were HER-2 FISH positive [8] .…”

Real-Time RT-PCR Analysis for Evaluating the Her2/neu Status in Breast Cancer

“…As discussed by Perreard et al [23] and Vinatzer et al [24] , the evaluation of Her2/neu mRNA expression did not show any statistically significant correlation with DNA amplification and protein expression. There was a clear overlap of expression levels of Her2/neu mRNA in the FISH and IHC subgroups, which could be explained on the heterogeneity of Her2/neu expression [25] . In spite of qRT-PCR representing an extremely sensitive and specific technique for determining transcript levels of Her2/neu [22] , cases with Her2/neu amplification and low Her2/neu mRNA levels were observed according to Gjerdrum et al [26] and Capizzi et al [27] .…”