Abstract P1-03-10: HER2 expression in clinical breast cancer samples: A novel detection methodology for HER2 protein quantitation using fluorescent nanoparticles

Abstract

Abstract Background: The human epidermal growth factor receptor-2 (HER2) is a member of a family of transmembrane tyrosine kinase receptors that play an important role in regulated normal cell growth and differentiation. The over-expression of HER2 in a subset of 15-20% of invasive breast cancers has an important bearing on prognosis, as HER2-positive tumors are associated with an aggressive clinical course and poor outcome. Targeting HER2-overexpression has been shown to be a remarkably effective therapeutic modality; however testing of tumor samples to assess the HER2 status of the patient's breast cancer is required. Clinical assays to assess the HER2 status in patients being considered for targeted therapy include immunohistochemistry (IHC), which detects protein over-expression, or fluorescence in situ hybridization (FISH), which detects gene amplification. Both the IHC and FISH methodologies have limitations. Given that the target of the currently approved drugs is the receptor protein, novel detections systems that could more accurately and quantitatively detect HER2 protein in clinical samples over a broad dynamic range would be advantageous and may be clinically helpful. Material and Methods: A novel detection technology using streptavidin-coated Phosphor Integrated Dot fluorescent nanoparticles (PID) has been developed that can be visualized by fluorescence microscopy and used for quantitative immunofluorescence detection of protein in clinical samples using computer assisted image analysis. In the current study, PID- nanoparticles were used to analyze HER2 protein expression in breast cancer cell lines and 120 well characterized breast cancer samples. These results have been compared with HER2 IHC and HER2 FISH analysis. Results: The expression levels of HER2 protein from 8 breast cancer cell lines was evaluated by antibody-binding capacity with FACS analysis. Formalin fixed paraffin embedded cell pellets for these cell lines were prepared and used for quantitative HER2 analysis by PID. The PID score/cell for each of these cell lines showed a strong linear correlation with antibody-binding capacity sites/cell by FACS analysis (R2 = 0.94). For the 120 breast cancer samples, PID score/cell was measured and compared against HER2 IHC membrane intensity measure by image analysis (Aperio) and HER2 FISH results. The HER2 PID score/cell showed a correlation coefficient of R2=0.72 versus the average HER2 copy number per cell by FISH, compared with a correlation coefficient of R2=0.41 for HER2 IHC membrane intensity measured by Aperio. For the HER2/CEP17 ratio, the correlation coefficient for the PID score/cell was R2=0.79 compared with a correlation coefficient of R2=0.32 for the HER2 IHC membrane intensity. Conclusions: PID-nanoparticles demonstrate great potential for the quantitative measurement of protein of clinical interest in routine clinical samples with morphologic confirmation of the tissue being studied. Further studies looking for PID-score thresholds for HER2 gene amplification and correlations with clinical outcome data are warranted and ongoing. Citation Format: Hicks DG, Goda H, Zhai H, Okada H, McMahon L, Sullivan N, Tang P, Nakano Y. HER2 expression in clinical breast cancer samples: A novel detection methodology for HER2 protein quantitation using fluorescent nanoparticles [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-03-10.