Abstract 49: Agreement of immunohistochemistry, fluorescence in situ hybridization, real-time quantitative polymerase-chain reaction, and quantitative reverse transcriptase PCR for Her-2/Neu status assessment in breast cancer patients: a single center, retrospective m
Authors: Zoppoli Gabriele , Garuti Anna , Rocco Ilaria , Palermo Claudia , Cirmena Gabriella , Carminati Enrico , Friedman Daniele , Verdun di Cantogno Ludovica , Sapino Anna , Patrone Franco , Ballestrero Alberto 
Published: August 2014

Abstract
Background: Immunohistochemistry (IHC) and fluorescence-in-situ hybridization (FISH) are the standard methods to assess human epidermal growth factor receptor 2 (HER-2) status in breast cancer (BC) patients. Real-time quantitative polymerase-chain-reaction (Q-PCR) and quantitative reverse transcriptase PCR (qRT-PCR) allow quantitative determination of gene copy number and gene expression on, respectively, DNA and RNA. These molecular methods are available to assess HER-2 amplification/overexpression but their use remains controversial. Here we performed a parallel comparison of these four methods to define their concordance rates and evaluate their relative role in HER-2 status determination. Patients and Methods: HER-2 status was determined by IHC, FISH, Q-PCR and qRT-PCR in a retrospectively assessed cohort of 130 BC patients. The studied set was enriched in cases scoring as 3+ by standard IHC analysis. Tests were performed blindly in parallel. Western blotting was performed on a subset of equivocal cases. Kappa statistics and ROC curves were used as appropriate, and concordance analyses were interpreted according to the ASCO/CAP guidelines. Results: Of the 130 enrolled patients, 47 (36%) were classified as positive and 27 (21%) as equivocal by IHC. With FISH, 50 patients (38%) were HER-2 amplified, while 80 (62%) were not. The overall agreement between FISH and Q-PCR was 98.5% (95% CI, 94.6% to 99.6%) with a k value of 0.97 (95% CI, 0.92 to 1). Assuming FISH as the standard reference, Q-PCR showed a sensitivity of 98% (95% CI, 89.5% to 99.6%) and a specificity of 98.8% (95% CI, 93.3% to 99.8%), with a global accuracy of 98%. The overall agreement between FISH and qRT-PCR was 96.9% (95% CI, 92.3% to 98.8%) with a k value of 0.94 (95% CI, 0.87 to 1). For both comparisons, the observed concordance values were greater than the 95% threshold required by the ASCO/CAP guidelines to validate novel approaches for HER-2 testing. 3% of samples showed HER-2 overexpression only by qRT-PCR analysis, in all cases belonging to the equivocal range as defined by either IHC or FISH. In these patients, WB confirmed higher HER-2 protein levels compared to healthy diploid controls. Conclusions: The high concordance between FISH and both Q-PCR and qRT-PCR supports the use of molecular tests as an alternative to current standard methods. Present results also suggest that qRT-PCR may be able to unequivocally classify patients belonging to the IHC/FISH equivocal range. Non-amplified HER-2 overexpressing BCs may account for the rare trastuzumab-responders observed in phase III trials, where trastuzumab response was assessed in HER-2-amplified vs. non-amplified cases.
Citation Format: Gabriele Zoppoli, Anna Garuti, Ilaria Rocco, Claudia Palermo, Gabriella Cirmena, Enrico Carminati, Daniele Friedman, Ludovica Verdun di Cantogno, Anna Sapino, Franco Patrone, Alberto Ballestrero. Agreement of immunohistochemistry, fluorescence in situ hybridization, real-time quantitative polymerase-chain reaction, and quantitative reverse transcriptase PCR for Her-2/Neu status assessment in breast cancer patients: a single center, retrospective m [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 49. doi:10.1158/1538-7445.AM2013-49