Tang, Cha‐Mei

Abstract Background: Renal cell carcinoma (RCC) is the most common type of kidney cancer responsible for ∼80% of kidney cancer cases. Circulating tumor cells (CTCs) are emerging as an important prognostic and predictive biomarker in several types of metastatic cancers. However, methods for CTC detection that target only EpCAM and/or cytokeratin (CK) to enrich CTCs, may fail to recognize the CTCs in clear cell RCC (ccRCC) because ccRCC lacks expression of EpCAM. The objective of this study was to develop a new technology capable of detecting CTCs including those with a mesenchymal phenotype in metastatic RCC. Methods: Blood samples from patients affected by metastatic RCC were provided by Mayo Clinic Arizona and analyzed by Creatv MicroTech. CellSieveTM microfilters, which have 7 µm diameter pores in a uniform array, with 160,000 pores in a 9 mm diameter area, were used for separation of CTCs in the blood specimen. 7.5 mL of whole blood was diluted in the prefixation buffer and filtered through the microfilter. Clinical immunohistochemical markers for RCC such as CD10 and vimentin were used to identify candidate CTCs. The cells collected on the filter were fixed, permeabilized, and stained with DAPI and fluorescent antibodies specific to CD10, vimentin, and CD45. The assay protocol was developed using three RCC cell lines, 786-O, Caki-1, and Caki-2, and validated using blood samples from patients with metastatic RCC. Results: The capture efficiencies for 786-O, Caki-1, and Caki-2 cell lines were determined to be 98%, 98% and 97%, respectively. On-filter antibody staining revealed heterogeneous expressions of vimentin and CD10 in RCC cells. Patient samples were collected in duplicate CellSaveTM tubes and processed one day later. A CD10+/vimentin+/CD45- cell population was detected in blood samples with enumeration ranging from 4 to 86 cells/7.5 mL of blood. We found that the RCC CTC profile was distinct from other types of cancers. The typical CTCs display abnormal morphology, including large nuclei (typically 15-30 µm in size), irregular cell size and shape, and high nucleus-to-cytoplasm ratio. Although CD10 and vimentin antibodies have cross-reactivity with white blood cells (WBC), the CTCs could be distinguished from WBC based on morphology, cell size and CD45- staining. Conclusions: We demonstrated that the CellSieveTM microfiltration assay is a very simple and efficient method to isolate a CD10+/vimentin+/CD45- cell population from patients affected by metastatic ccRCC. Future studies include fluorescence in situ hybridization to confirm loss of 3p, a molecular event that occurs in >90% of ccRCC. We are also analyzing blood samples from patients treated with tyrosine kinase inhibitors to determine if cell enumeration is predictive of response to treatment. This technology might greatly facilitate detection of EpCAM negative ccRCC CTCs. The clinical significance of these unusual, large, rod-shaped, naked nuclei require further molecular characterization. Citation Format: Peixuan Zhu, Thai Ho, Erik P. Castle, Richard W. Joseph, Melissa L. Stanton, Shuhong Li, Daniel Adams, Olga V. Makarova, Platte T. Amstutz, Cha-Mei Tang. Isolation and identification of circulating tumor cells in renal cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3081. doi:10.1158/1538-7445.AM2014-3081

AbstractRecent studies reporting hundreds, to thousands, of circulating tumor cells (CTCs) in the blood of cancer patients have raised questions regarding the prevalence of CTCs, as enumerated by the CellSearch® CTC Test. Although CellSearch has been shown to consistently detect clinically relevant CTCs; the ability to only capture EpCAM positive cells has led to speculation that it captures limited subsets of CTCs. In contrast, alternative approaches to CTC isolation are often cited as capturing large numbers of CTCs from patient blood. Not surprisingly the number of cells isolated by alternative approaches show poor correlations when compared to CellSearch, even when accounting for EpCAM presence or absence. In an effort to address this discrepancy, we ran an exploratory method comparison study to characterize and compare the CTC subgroups captured from duplicate blood samples from 30 breast and prostate cancer patients using a microfiltration system (CellSieve™) and CellSearch. We then categorized the CellSieve Cytokeratin(CK)+/CD45−/DAPI+ cells into five morphologically distinct subpopulations for correlative analysis. Like other filtration techniques, CellSieve isolated greater numbers of CK+/CD45− cells than CellSearch. Furthermore, analysis showed low correlation between the total CK+/CD45− cells captured by these two assays, regardless of EpCAM presence. However, subgrouping of CK+/CD45−/DAPI+ cells based on distinct cytokeratin staining patterns and nuclear morphologies elucidated a subpopulation correlative to CellSearch. Using method comparison analyses, we identified a specific CTC morphology which is highly correlative between two distinct capture methods. These data suggests that although various morphologic CTCs with similar phenotypic expressions are present in the blood of cancer patients, the clinically relevant cells isolated by CellSearch can potentially be identified using non‐EpCAM dependent isolation. © 2014 The Authors. Published by Wiley Periodicals, Inc.