Abstract 1553: Redefining CTCs: Detection of cytokeratin-negative circulating tumor cells (CTCs)
Authors: Pecot Chad V. , Bischoff Farideh , Lin Yvonne , Jaladurgam Padmavathi , Merritt William , Pircher Tony , Mikolajczyk Steve , Mayer Julie , Wong Karina , Pham Tam , Bottsford-Miller Justin , Stone Rebecca , Celestino Joseph , Nick Alpa , Eng Cathy , Sood Anil 
Published: June 2012

Abstract
Introduction: There is growing evidence that CTCs display an epithelial-mesenchymal transition phenotype. Current enrichment techniques rely upon epithelial markers for capture (anti-EpCAM antibodies and cytokeratin [CK]) and may miss important populations of circulating tumor cells. We sought to develop a new method for identification of CTCs.
Methods: After IRB approval, blood from patients with ovarian or colorectal carcinoma was collected. Cell Enrichment and Extraction (CEE) technology, a microfluidic-based device using an antibody cocktail targeting epithelial and mesenchymal markers, was utilized for the capture and analysis of rare cells in blood. We enumerated cells that were CK+ and/or contained complex aneuploidy by fluorescent in situ hybridization (FISH). Blood samples from healthy volunteers served as a negative control. Fresh frozen tumor samples were analyzed on a subset of patients to search for concordance of molecular alterations with CTCs. An orthotopic ovarian cancer model was used to evaluate effects of chemotherapy on CTCs.
Results: Enumeration of CK+ cells identified an average of 1 and 2 cells per 10 mL blood in ovarian and colorectal cancer patients, independent of stage or tumor grade. Enumeration did not correlate with serum tumor marker levels. The majority of CK+ cells had complex aneuploidy. Ovarian cancer patients had equal numbers of CK- and CK+ complex aneuploid cells (p=1.0). A three-fold increase in CK- over CK+ complex aneuploid cells was noted in patients with colorectal cancer, although not statistically significant (p&lt;0.19). The total number of complex aneuploid cells did not correlate with serum tumor markers. Similar patterns of complex aneuploidy were identified in patients’ primary tumors as were seen in their CK- cells from blood, indicating that the cells identified in circulation were indeed CTCs. No CK+ or complex aneuploid cells were found in healthy volunteers. In the orthotopic mouse model, compared to tumor-bearing controls, chemotherapy treatment initially lead to a 17-fold increase in apoptotic appearing CTCs (p&lt;0.02) followed by a 15-fold decrease in CTCs (p&lt;0.03), which also reflected tumor response.
Discussion: Here, we have developed and characterized a novel and robust method for detection of CTCs. The power of this approach lies in its ability to detect CTCs with either an epithelial or EMT-positive phenotype. Our findings suggest current enrichment techniques may be missing an important population of cells. Complex aneuploid CK- cells challenge the current definition of a CTC and merit further development in clinical settings.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1553. doi:10.1158/1538-7445.AM2011-1553