Her2 assessment using quantitative reverse transcriptase polymerase chain reaction reliably identifies Her2 overexpression without amplification in breast cancer cases

Abstract

Background: Immunohistochemistry (IHC) and fluorescent-in situ hybridization (FISH) are standard methods to assess human epidermal growth factor receptor 2 (HER2) status in breast cancer (BC) patients. Real-time quantitative polymerase-chain-reaction (qRT-PCR) is able to detect HER2 overexpression. Here we compared FISH, IHC, quantitative PCR (qPCR), and qRT-PCR to determine the concordance rates and evaluate their relative roles in HER2 determination.

Patients and methods: We determined HER2 status in 153 BC patients, using IHC, FISH, Q-PCR and qRT-PCR. In discordant cases, we directly measured HER2 protein levels using Western blotting.

Results: The overall agreement (OA) between FISH and Q-PCR was 94.1, with a k value of 0.87. Assuming FISH as the standard reference, Q-PCR showed an 86.1% sensitivity and a 99.0% specificity with a global accuracy of 91.6%. OA between FISH and qRT-PCR was 90.8% with a k value of 0.81. Of interest, the disagreement between FISH and qRT-PCR was mostly restricted to equivocal cases. HER2 protein analysis suggested that qRT-PCR correlates better than FISH with HER2 protein levels, particularly where FISH fails to provide conclusive results.

Significance: qRT-PCR may outperform FISH in identifying patients overexpressing HER2 protein. Q-PCR cannot be used for HER2 status assessment, due to its suboptimal level of agreement with FISH. Both FISH and Q-PCR may be less accurate than qRT-PCR as surrogates of HER2 protein determination.

Fig. 1

ROC curves for Q-PCR (a) and Q-RT-PCR (b) compared to FISH in non-equivocal cases. The blue and green squares represent the corrected partial areas under the curve (pAUC) for the graphic regions encompassing the 100–95% specificity and sensitivity areas, respectively. The value on the topleft corner of each panel indicates the optimal cutoff, with specificity and sensitivity in parentheses

Fig. 2

Dilution effect by not-HER2 amplified cells was analyzed in serial dilutions of HER2-positive SKBR3 cell line with HER2-negative MCF10A cell line. Columns represent the mean of three independent experiment performed by Q-PCR (a) and qRT-PCR (b). Copy number and gene expression were measured according to the 2^−[ΔΔCt] algorithm. SKBR3 is a mammary carcinoma cell line with a medium–high level of HER2 amplification. HER2 human epidermal growth factor receptor 2, Q-PCR quantitative polymerase-chain-reaction, qRT-PCR quantitative reverse transcriptase polymerase-chain-reaction