Abstract 2422: Isolation of canonical and non-canonical circulating tumor cells (CTCs) by negative depletion using the Harpoon CTC Isolator and Chip
Authors: Chianese David , Connelly Mark , Morano Carrie , Bui Thai , Gross Steven , Sanders Renouard , Barber Tom , Kapur Ravi , Maheswaran Shyamala , Toner Mehmet , Haber Daniel 
Published: July 2016

Abstract
Today, circulating tumor cells (CTCs) can provide important prognostic information about a patient. However, to one day help guide decisions for precision medicine, more and increasingly sophisticated information will have to be extracted from each patient blood sample. We describe a new microfluidic CTC isolation technology, the Harpoon CTC Isolator and Chip (Hrp Isolator). From a single sample the Hrp Isolator can enable the analysis of plasma components such as ctDNA, as well as the ability to enrich all circulating tumor cell types, regardless of their antigenic expression. To enrich the CTCs from 7.5 mL of whole blood, the Hrp Isolator removes the red blood cells and platelets based on their small size and the white blood cells by negative depletion using magnetic particles. The time required to process 7.5 mLs of blood is approximately 90 minutes. In contrast, the CELLSEARCH® CTC Test (CS), the only US FDA-cleared test, uses antigens expressed by CTCs to target the CTCs and positively select them out of blood. To enumerate CTCs from patient samples, the output from the Hrp Isolator was then loaded into and run on the CELLSEARCH® System using the CS test (Hybrid method). CTCs were enumerated from 37 breast and prostate cancer patients by CS and by the Hybrid method. At least 1 CTC was found in 23 (62%) patient samples by the Hybrid method versus 17 (46%) by CS. The product of the Hrp Isolator enrichment contained on average the same numbers of CTCs as were found by CS (mean = 13.3 ± 18 by CS versus 12.0 ± 16 by Hybrid) in patient samples having &lt;75 CTCs. For samples with high CTC counts (&gt;75), the Hybrid method may have underestimated the total Hrp Isolator CTC count. An important feature of the Hrp Isolator is its ability to enrich CTCs that are negative or low expressers of epithelial cell adhesion molecule (EpCAM) or cytokeratin (CK). These cells could have been missed by positive-selection technologies such as CS and may not have been counted using the Hybrid method. CTC spiked samples were run on the Hrp Isolator and the CTC enriched product was spun onto microscope slides, stained, and examined using a Vectra Multispectral Imaging System (Hrp+Vec method). Using the Hrp+Vec method, 79% to 100% of spiked CTCs were recovered depending on the slide fixation method used. Also, using Hrp+Vec we confirmed the ability of the Harpoon Isolator to enrich EpCAM-/CK+ and EpCAM+/CK- CTCs from patient samples. This establishes the existence of “non-canonical” tumor cell types in circulation and adds to the biological diversity of the CTC population in circulation. These results emphasize the need to further characterize all tumor cell populations and to determine what, if any, clinical significance may be assigned to these diverse CTC types.
Citation Format: David Chianese, Mark Connelly, Carrie Morano, Thai Bui, Steven Gross, Renouard Sanders, Tom Barber, Ravi Kapur, Shyamala Maheswaran, Mehmet Toner, Daniel Haber. Isolation of canonical and non-canonical circulating tumor cells (CTCs) by negative depletion using the Harpoon CTC Isolator and Chip. [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 2422.