Abstract 3149: Enumeration and mutational profiling of CTCs and comparison to ctDNA and colorectal cancer liver metastases
Authors: Kidess-Sigal Evelyn , Liu Haiyan E. , Triboulet Melanie , Che James , Poultsides Georges A. , Visser Brendan C. , Marziali Andre , Lee Marc , Vysotskaia Valentina , Wiggin Matthew , Ramani Vishnu C. , Keilholz Ulrich , Tinhofer Ingeborg , Zia Amin , Coller John , Norton Jeffrey A. , Sollier Elodie , Jeffrey Stefanie S. 
Published: July 2016

Abstract
Background
Colorectal cancer (CRC) is the 3rd most common cancer diagnosed worldwide in both men and women. Only 39% of cancers are diagnosed at a localized stage, and 5-year survival rates decrease rapidly for patients with advanced and metastasized disease (stage III 61%, stage IV 8%). Better markers for detection of disease progression, therapeutic resistance and minimal residual disease are still needed. Liquid biopsies, such as CTCs and ctDNA, are emerging biomarkers shed by the tumor into the blood stream. Both markers currently are attracting growing interest for their use in disease prognosis, early detection of recurrence and are promising candidates for guiding cancer therapy in real-time.
Method
For rapid label-free isolation of CTCs from peripheral blood we used the Vortex technology, a microfluidic device using inertia and laminar microvortices. From 15 patients with metastatic CRC to the liver that underwent liver metastatectomy with curative intent, we collected CTCs preoperatively, at the 5th postoperative day and during follow-up visits. Cells collected were immunostained for EpCAM, CD45 and DAPI, enumerated using standardized classification criteria, and subjected to Sanger sequencing. CTC enumeration and mutational patterns were compared to the primary tumor, liver metastases and ctDNA (detected by a multiplexed PCR and enrichment technology; Kidess E et al., 2015) as well as CEA levels when available.
Results
41 blood samples from 15 patients were collected at different time points prior to and after surgical resection of liver metastases. More CTCs were found in preoperatively collected CRC patient samples (2.4 CTCs/mL, 0.1 - 5.5/mL) than in age-matched healthy controls (0.1 CTCs/mL, 0 - 0.4/mL). 80% of all CRC samples were identified as positive for CTCs (based on a calculated threshold from healthy controls), with varying levels of EpCAM expression (81.4% of CTCs being EpCAM+). The number of CTCs for each patient, showed a close correlation to clinical parameters and ctDNA levels: detection of CTCs, CTC mutational profiles as well as ctDNA revealed minimal residual disease and anticipated tumor recurrence earlier than carcinoembryonic antigen (CEA) value or imaging. For example, for P006, postoperative imaging surveillance revealed progressive disease, which was accompanied by rising levels of CTCs (up to 29 CTCs/mL at the last time point) and PIK3CA mutant DNA in both plasma ctDNA and CTC DNA, while CEA remained in the normal range.
Conclusion
Our data illustrate that CTCs as well as ctDNA can efficiently reveal disease recurrence as well as disease progression earlier than imaging and far more reliable compared to CEA, the currently standard biomarker for CRC. Beyond enumeration, CTC molecular analysis gives additional information and will potentially help to promote the development of tailored therapies for every individual patient.
Citation Format: Evelyn Kidess-Sigal, Haiyan E. Liu, Melanie Triboulet, James Che, Georges A. Poultsides, Brendan C. Visser, Andre Marziali, Marc Lee, Valentina Vysotskaia, Matthew Wiggin, Vishnu C. Ramani, Ulrich Keilholz, Ingeborg Tinhofer, Amin Zia, John Coller, Jeffrey A. Norton, Elodie Sollier, Stefanie S. Jeffrey. Enumeration and mutational profiling of CTCs and comparison to ctDNA and colorectal cancer liver metastases. [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 3149.