Abstract 4530: Single HER2-positive tumor cells are detected in initially HER2-negative breast carcinomas using the DEPArray™-HER2 FISH workflow
Authors: Koenig Lisa , Gerber Amanda , Khurana Aditi , Bischoff Farideh Z. , Kasimir-Bauer Sabine 
Published: August 2018

Abstract
Background: In breast cancer (BC), HER2 expression does not only differ within various primary tumor (PT) areas, but also with respect to the expression on circulating tumor cells (CTCs). Despite clinical guidelines defined by CAP/ASCO, PT HER2 analysis displays limitations due to pre-analytical sampling, methodology and intra-tumor heterogeneity. Fluorescence in situ hybridization (FISH) is used for analyzing HER2/neu (ERBB2) gene amplification and for re-solving inconclusive HER2 immunohistochemistry results. In this study, we aimed to evaluate BC intra-tumor heterogeneity using the DEPArray™ - HER2 FISH workflow in BC patients characterized as i) PT HER2 neg / CTC HER2/neu pos (n=25).
Methods: 50 µm FFPE tumor curls (n=25) were cut, deparaffinized and underwent antigen retrieval. After dissociation into a single cell suspension, the cells were stained for cytokeratin (CK-AF488), vimentin (Vim-AF647) and DAPI to distinguish between stroma and tumor cells. These two cell populations were separated using the DEPArray™, an image-based single cell sorting system. Samples containing ~ 200 intact CK+/Vim-/DAPI+ tumor cells were deemed suitable for pure tumor cell recovery. Subsequently, HER2 FISH analysis was performed on the recovered single cell tumor sample for HER2 and chromosome 17 signal evaluations using a dual HER2/CC17 probe. The results obtained with the DEPArray™ - HER2 FISH workflow were then compared with the HER2 status obtained by routine pathology and correlated with the HER2 status on CTCs, enriched from 10 ml blood using positive immunomagnetic selection followed by RNA isolation and subsequent gene expression analysis by reverse transcription and Multiplex-PCR (AdnaTest, Qiagen).
Results: A concordant result for HER2 analysis comparing routine pathology and DEPArray™ - HER2 FISH analysis was found for 84% (21/25) of the patients. A discordant result was identified in 4/25 patients using the DEPArray™ - HER2 FISH workflow. 2/25 BC patients were proven to be HER2 positive in our approach, despite being tested HER2 negative in routine pathology. Two other patients showed an equivocal HER2 status in the DEPArray™ - HER2 FISH workflow, but a negative result in routine pathology. Consequently, four BC patients would have been eligible for anti-HER2 treatment.
Conclusion: The DEPArray™ system allows the recovery of a pure tumor cell fraction for subsequent HER2 FISH analysis and shows a high concordance with the HER2 status performed by routine pathology. However, the DEPArray™ - HER2 FISH workflow offers a more precise and accurate diagnostic test and could be used for BC patients at high risk, e.g. triple negative BC patients, patients with HER2/HER3 positive CTCs or in HER2 inconclusive BC cases to offer anti-HER2 treatment.
Citation Format: Lisa Koenig, Amanda Gerber, Aditi Khurana, Farideh Z. Bischoff, Sabine Kasimir-Bauer. Single HER2-positive tumor cells are detected in initially HER2-negative breast carcinomas using the DEPArray™-HER2 FISH workflow [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4530.