Abstract P6-01-01: Immunohistochemical staining and in vitro analysis of HER2-positive breast cancer using trastuzumab and pertuzumab to develop an appropriate tracer in image-guided surgery

Background: Pre- and intraoperative visualization of cancer cells using monoclonal antibody-based multimodal tracers can demarcate tumor margins in breast-conserving surgery. We focused on trastuzumab (Tmab) to develop a tracer for HER2-positive breast cancer by conjugation with a fluorescent dye. However, whether Tmab can be used as an imaging tracer for patients who receive Tmab as neoadjuvant therapy (NAD) is unclear, because tumor-cell HER2 could be bound by NAD Tmab at the time of surgery. This study evaluated immunohistochemical (IHC) staining and in vitro experiments with Tmab and pertuzumab (Pmab) as primary antibodies to find a suitable tracer. Pmab has a different antigen epitope than Tmab. Methods: We included 43 patients with HER2-positive breast cancer, who were treated between 2010 and 2016. The NAD cohort (n=10, with 26 lesions) received chemotherapy and Tmab before surgery. The Tmab-naive cohort (n=33, with 95 lesions) did not receive chemotherapy or Tmab before surgery. We excluded NAD patients with pathological complete responses. We evaluated the lesions, using IHC with Tmab and Pmab. We also performed flow cytometry and fluorescent microscopic analysis (FMA) using Tmab and Pmab conjugated with a fluorescent agent for the HER2-negative MCF7 and HER2-positive HCC1954 cell lines, which were pre-treated with Tmab to model NAD conditions. Results: IHC with anti-HER2 antibody showed positive staining in all patients. Tmab staining was less intense, but positive Tmab IHC reactions were detected on tumor cell membranes in 77.8% of lesions in the naive cohort and 70.8% in the NAD cohort. Pmab staining was seen in 46.3% of naive cohort lesions and 22.2% of NAD lesions. Notably, we observed cytoplasmic staining in 87.8% of Pmab-negative cases in the NAD cohort. Flow cytometry showed less Tmab binding than Pmab binding in HCC1954 cells. After 24 hours9 pretreatment incubation with Tmab, FMA showed a clear decrease in Tmab binding from 73.3% (without pretreatment) to 5.7%(after pretreatment), and a smaller decrease in Pmab binding from 72.7% (without pretreatment) to 66.4% (after pretreatment) in HCC1954 cells. Conclusions:Pmab might be a suitable tracer for image-guided surgery after NAD, but identifying a suitable tracer for HER2-positive breast cancer will require further study. Citation Format: Otsubo R, Kuppen PJ, Bhairosingh S, Vahrmeijer AL, Smit VT, Nagayasu T, van de Velde CJ, Sier CF. Immunohistochemical staining and in vitro analysis of HER2-positive breast cancer using trastuzumab and pertuzumab to develop an appropriate tracer in image-guided surgery [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-01-01.