Abstract 3159: Identification of circulating tumor cells from renal cell carcinoma patients by a multi-parameter flow cytometry assay
Authors: Desotelle Joshua A. , Pak Chorom , Heninger Erika , Schehr Jennifer L. , McKay Rana R. , Gibbs Benjamin K. , Norton Craig , Choueiri Toni K. , Lang Joshua M. 
Published: July 2016

Abstract
BACKGROUND: Renal cell carcinoma (RCC) is one of the top ten causes of cancer death in the United States. The last ten years have shown a dramatic increase in the number of available treatment options, however metastatic RCC remains largely incurable. The classes of drugs that have been developed can be divided into agents that target the Vascular Endothelial Growth Factor (VEGF) pathway (Sunitinib, Sorafenib, Pazopanib, Axitinib, Bevacizumab), those that target the mammalian Target of Rapamycin (mTOR) pathway (Everolimus, Temsirolimus) and immune based therapies (IL-2 and PD-1 inhibition). There are currently limited biomarkers to guide clinical decisions, mainly due to the lack of tumor cells for longitudinal molecular analysis. Circulating tumor cells (CTCs) are potential a source of tumor cells that can be identified from a blood draw for serial analysis. CTCs have not been reliably detected in RCC due to the significant heterogeneity and high rate of false positive events in this disease when EpCAM has been used. We sought to identify RCC CTCs with alternative markers, such as carbonic anhydrase IX (CAIX) which is found in greater than 90% of clear cell RCC tumors.
METHODS AND RESULTS: To evaluate for the presence and subtypes of CTCs from patients with RCC, we utilized a multi-parametric flow cytometry assay. We evaluated heterogeneity across subpopulations of putative CTCs with Epithelial Cell Adhesion Molecule (EpCAM), Carbonic Anhydrase IX (CAIX), Carbonic Anhydrase XII (CAXII), PAX8, and Cytokeratin (CK). Negative controls for immune and endothelial events were performed with markers for CD45, CD14, CD34, CD11b and CD61. We tested twenty blood samples from patients with RCC at the Dana Farber Cancer Institute and University of Wisconsin Carbone Cancer Center. CTC frequency in RCC ranges from 0-5410 CAIX+/CK+ events with a median of 24.5 putative CTCs/7.5mL of blood. A subset of patients with radiographic progression had a higher number of CTCs with a median of 295.2 CTCs/7.5 mL. A range of 1-231 EpCAM+/CK+ events were identified with a median of 5.5 CTCs/7.5mL. There was low frequency of events being CAIX+/EpCAM+/CK+, with a median of 1 CTC/7.5mL of blood. Assay specificity was dramatically improved through the combination of multiple positive markers with stains for immune and endothelial cells given frequent non-specific staining for cytokeratin in RCC blood samples.
CONCLUSIONS: CTCs can be identified in patients with RCC using non-traditional markers. CAIX is a more sensitive marker than EpCAM to identify putative CTCs from patients with RCC. Specificity in the assay is critical given the high frequency of false positive events identified if only CD45 is used as a marker for immune cells. Our investigation is ongoing for further molecular characterization of orthogonal endpoints in identified CTCs. Future directions include longitudinal monitoring of CTCs during treatment with VEGF inhibitors.
Citation Format: Joshua A. Desotelle, Chorom Pak, Erika Heninger, Jennifer L. Schehr, Rana R. McKay, Benjamin K. Gibbs, Craig Norton, Toni K. Choueiri, Joshua M. Lang. Identification of circulating tumor cells from renal cell carcinoma patients by a multi-parameter flow cytometry assay. [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 3159.