Abstract 4585: Development of multi-marker capture and analysis of circulating tumor cells in renal cell carcinoma
Authors: Bade Rory M. , Gibbs Benjamin K. , Sperger Jamie M. , Kyriakopolous Christos , Emamekhoo Hamid , McKay Rana R. , Choueiri Toni K. , Lang Joshua M. 
Published: August 2018

Abstract
Purpose: Circulating biomarkers are an emerging tool to monitor treatment response and the emergence of resistant phenotypes. However, studies of circulating biomarkers, including circulating tumor cells (CTCs), in patients with clear cell renal cell carcinoma (ccRCC) have been limited due to difficulty in biomarker identification. Platforms relying on EpCAM and cytokeratin to identify CTCs have been limited due to significant phenotypic and intrapatient heterogeneity in renal cancer. Carbonic anhydrase IX (CAIX) and XII (CAXII) are more broadly expressed in ccRCC and recently been shown to capture CTCs from patients with ccRCC. However, downregulation of these targets can also occur; EpCAM is also expressed on a subset of cells that could go undetected if only CAIX was used to capture these cells. The aim of this study is to optimize multi-marker capture and analysis of ccRCC CTCs using EpCAM, CAIX and CAXII for further molecular analysis.
Methods: We utilized the VERSA platform, an integrated CTC capture and analysis technology, to optimize capture of multiple ccRCC cell lines using antibodies to CAIX and/or EpCAM. To maximize the capture efficiency of ccRCC CTCs, we altered the magnetic particle type, antibody concentration, and tested both direct and indirect capture methods. Once an optimal method of capture was determined, we captured CTCs in an initial cohort of ten patients with ccRCC. CTCs were identified as cells that were captured by either CAIX or EpCAM, had an intact nucleus, were negative for CD45/CD34/CD66b, and positive for cytokeratin.
Results: Capture of cell lines show that a combined CAIX + EpCAM capture was more efficient than single antibody capture using either EpCAM or CAIX alone. The type of magnetic particle used in the assay also affected capture efficiency. Sera-Mag beads (GE Healthcare) captured significantly more cells than FlowComp Dynabeads (Life Technologies) (94% vs 76%). Further increases in efficiency were made by incubating with antibody prior to bead conjugation (indirect binding, 94%) when compared to incubating the cells with antibody-conjugated beads (direct binding, 98%). This may result from increased accessibility of free antibody to partially obstructed antigens that is unique to renal cell carcinoma. This optimized assay has now been applied to ccRCC patients and has identified CTCs in up to 90% of patients with metastatic disease.
Conclusions: We have increased the capture efficiency and identification of ccRCC cells by capturing with a combination of CAIX and EpCAM antibodies and optimizing bead and binding conditions. In doing so, we are able to identify and interrogate populations of CTCs that would be lost in capture methods that rely on EpCAM alone. These assays are now being utilized in multiple biomarker and therapeutic trials for patients with clear cell renal cell carcinoma.
Citation Format: Rory M. Bade, Benjamin K. Gibbs, Jamie M. Sperger, Christos Kyriakopolous, Hamid Emamekhoo, Rana R. McKay, Toni K. Choueiri, Joshua M. Lang. Development of multi-marker capture and analysis of circulating tumor cells in renal cell carcinoma [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 4585.