Body: Guidelines worldwide focus on the importance of precise, reproducible, and quality assurance of Fluorescent In Situ Hybridization (FISH) methods for testing companion diagnostic markers, including Human Epidermal Growth Factor Receptor 2 (HER2) gene amplification in breast cancer. Despite these guidelines, variations in test results due to pre-analytical sampling and tissue processing are observed. In this study, we demonstrate a unique approach to isolating pure and intact tumor cells from breast cancer Formalin-Fixed, Paraffin-Embedded (FFPE) samples for precise subsequent FISH analysis.
Methods: Fifty-micron thick FFPE curls from both HER2 non-amplified breast cancer tumors (n = 4; each with a reported HER2/CEP17 ratio 1.8) and positive control SKBr3 breast cancer cells were tested. Isolation of ∼250 pure and intact cytokeratin-positive/vimentin-negative/DAPI positive tumor cells from each sample was achieved using the DEPArray™ platform, an automated system enabling image-based cell sorting with single-cell resolution for pure cell population isolation and collection. Recovered cells were then cyto-spun onto poly-L coated glass slides prior to standard dual-color HER2/CEP17 FISH (Path Vysion Abbott/Vysis) analysis.
Results: Positive HER2 amplification levels for the FFPE derived control SKBr3 cells were observed (HER2/CEP17 ratio >4.4) and consistent with levels reported in the literature. Among the patient samples, ≥75% of the DEPArray™ isolated tumor cells were recovered onto slides prior to FISH. Through routine FISH scoring, an expected non-amplified result was observed for each patient sample, with observed HER2/CEP17 ratios only ranging from 1.1 to 1.4.
Conclusion: We demonstrate feasibility in performing FISH for HER2/CEP17 on pure and intact tumor cells isolated from breast cancer derived FFPE using the DEPArray™ platform. Using a 50-micron section permitted recovery of whole, intact, tumor cells based on immunostaining for cytokeratin, vimentin, and DAPI. Efficient recovery of the DEPArray™ sorted cells onto slides further permitted routine FISH analysis of only tumor cells. These preliminary results imply the possibility of more precise FISH analysis when standard FISH results are inconclusive or when insufficient tumor content prohibits downstream analysis. Evaluation of larger numbers of patient samples is underway.
Citation Format: Amanda Gerber, Trisky Clarin, Ambica Bhandari, Valeria Sero, Chiara Bolognesi, Gianni Medoro, Nicolò Manaresi, Mathew Moore, Philip D. Cotter, Farideh Bischoff. Isolation and analysis of pure intact tumor cell populations from FFPE: Implications for more precise HER2 FISH testing in breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1374.