Abstract
Abstract Introduction: Isolation of circulating tumor cells based on cell surface markers is often hindered by epithelial to mesenchymal transition with loss of epithelial antigens. Low numbers of CTCs and fewer cases of CTC positive pancreatic cancer compared to other cancers (e.g. breast cancer) lead to the assumption that pancreatic tumors do not release CTCs as frequently or that the pancreatic CTCs lack EpCAM expression. Isolation of CTCs based on their size is independent of EMT-like phenotypical changes. We therefore compared a filtration-based isolation method with an EpCAM-based isolation method. We optimized CTC detection by using a highly sensitive anti-cytokeratin antibody panel for the detection of cancer cells with moderate cytokeratin expression. In order to use isolated CTCs as liquid biopsy for tumor characterization and treatment selection, downstream analysis is necessary. We here show the feasibility of mutational analysis of isolated CTCs by castPCR. Methods: EpCAM high, medium and low cells were enriched and isolated by an EpCAM-based immunomagnetic procedure (IsoFlux) and a filtration device (Siemens) to determine recovery rates of both methods. Isolated cells were detected by an improved immunofluorescent staining with an anti-Cytokeratins, anti-EpCAM and anti-PBMC panel and characterized by competitive allele-specific TaqMan PCR (castPCR) for KRAS mutations. Results: Cytokeratin expression is crucial for detection of CTCs in a high background of blood cells. Improvement of the staining protocol helps to increase the sensitivity of detection. The filtration based approach is superior to the surface antigen-based isolation. With the size-dependent method we obtained a recovery rate of 52 % even for EpCAM-low cells compared to only 1 % based on the immune-affinity purification. Cells isolated by filtration can be characterized for expression of therapeutic targets by immunostaining. The utility of the size dependent platform for subsequent functional characterization of the CTCs was also demonstrated by detection of k-ras mutations in single isolated CTCs by castPCR. Conclusion: For CTCs undergoing EMT, filtration yields higher recovery compared to the standard surface antigen (EpCAM)-based methods. Isolation by filtration also allows for mutational analysis which can be used to confirm the identity of the isolated cell as CTC. In addition, mutational analysis of CTCs can be used to guide the treatment of patients. Use of liquid biopsies for treatment selection will facilitate truly personalized medicine. Citation Format: Oliver von Ahsen, Nora Brychta, Thomas Krahn. Improved isolation and detection of circulating tumor cells of pancreatic cancer patients with characterization by mutational analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3792. doi:10.1158/1538-7445.AM2017-3792
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