Capture, Isolation, And Mutational Analysis Of Single Pancreatic Circulating Tumor Cells Using Nanovelcro Technology

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Introduction: Circulating tumor cells (CTCs) have been developed as a biomarker in several cancers, but not in pancreatic cancer (PDAC). The available clinical technology for CTC study, CellSearch, suffers from limited ability to perform molecular analysis. We sought to evaluate a novel microfluidic NanoVelcro technology coupled to laser micro-dissection (LMD), for CTC capture and single cell analysis of KRAS mutations to confirm tumor origin of captured CTCs. Methods: NanoVelcro utilizes a nano-spun polymer fiber, modified with streptavidin, to capture CTCs using biotinylated antibodies. The system was optimized with the CFPAC-1 (EpCAM+, KRAS G12 to V) cell line spiked into healthy donor blood. Patient samples were collected from 2 mL peripheral venous blood and run at the optimized flow rate of 1mL/hr. CTCs were defined by size (≥ 10 µm) and immuno-staining pattern (DAPI+/CK+/CD45-). Using LMD, single cells were isolated and subjected to whole genome amplification followed by PCR of KRAS exon 1 and subsequent Sanger Sequencing. Results: The NanoVelcro system allowed for detection of mutant KRAS in isolated CFPAC-1 cells. 6 CTCs were then isolated from a known KRAS G12 to V mutant patient and 3/6 (50.0%) captured CTCs were found to have KRAS G12 to V mutations while normal WBCs from the same patient contained wild-type KRAS. Conclusions: The microfluidic NanoVelcro technology demonstrated capture of pancreatic CTCs and may prove useful in the development of CTCs as a biomarker in PDAC. Application of LMD enabled single cell KRAS mutational analysis confirming cancer origin of isolated CTCs and supporting our current PDAC CTC identification criteria based on immuno-staining. This promising technology, combined with LMD, opens the door for molecular characterization of CTCs, thus providing insight into the biology of metastasis while potentially revealing molecular targets for therapeutics. Citation Format: Jacob S. Ankeny, Shuang Hou, Millicent Lin, Matthew Frias, Hank OuYang, Min Song, Matthew M. Rochefort, Mark D. Girgis, Hsian-Rong Tseng, James S. Tomlinson. Capture, isolation, and mutational analysis of single pancreatic circulating tumor cells using NanoVelcro technology. [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 3070. doi:10.1158/1538-7445.AM2014-3070